Compositions and methods for use in recombinational cloning of nucleic acids

ABSTRACT

The present invention relates generally to compositions and methods for use in recombinational cloning of nucleic acid molecules. In particular, the invention relates to nucleic acid molecules encoding one or more recombination sites or portions thereof, to nucleic acid molecules comprising one or more of these recombination site nucleotide sequences and optionally comprising one or more additional physical or functional nucleotide sequences. The invention also relates to vectors comprising the nucleic acid molecules of the invention, to host cells comprising the vectors or nucleic acid molecules of the invention, to methods of producing polypeptides using the nucleic acid molecules of the invention, and to polypeptides encoded by these nucleic acid molecules or produced by the methods of the invention. The invention also relates to antibodies that bind to one or more polypeptides of the invention or epitopes thereof. The invention also relates to the use of these compositions in methods for recombinational cloning of nucleic acids, in vitro and in vivo, to provide chimeric DNA molecules that have particular characteristics and/or DNA segments.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of the filing dates of U.S.Provisional Application Nos. 60/122,389, filed Mar. 2, 1999, 60/126,049,filed Mar. 23, 1999, and 60/136,7844, filed May 28, 1999. The presentapplication is also related to U.S. application Ser. No. 08/486,139,filed Jun. 7, 1995 (now abandoned), Ser. No. 08/663,002, filed Jun. 7,1996 (now U.S. Pat. No. 5,888,732), Ser. No. 09/005,476, filed Jan. 12,1998, Ser. No. 09/177,387, filed Oct. 23, 1998, Ser. No. 09/233,492,filed Jan. 20, 1999, Ser. No. 09/233,493, filed Jan. 20, 1999, Ser. No.09/296,280, filed Apr. 22, 1999, Ser. No. 09/296,281, filed Apr. 22,1999, Ser. No. 09/432,085, filed Nov. 2, 1999, and Ser. No. 09/438,358,filed Nov. 12, 1999. The disclosures of all of the applicationscross-referenced above are incorporated by reference herein in theirentireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to recombinant DNA technology.More particularly, the present invention relates to compositions andmethods for use in recombinational cloning of nucleic acid molecules.The invention relates specifically to nucleic acid molecules encodingone or more recombination sites or one or more partial recombinationsites, particularly attB, attP, attL, and attR, and fragments, mutants,variants and derivatives thereof. The invention also relates to suchnucleic acid molecules wherein the one or more recombination sitenucleotide sequences is operably linked to the one or more additionalphysical or functional nucleotide sequences. The invention also relatesto vectors comprising the nucleic acid molecules of the invention, tohost cells comprising the vectors or nucleic acid molecules of theinvention, to methods of producing polypeptides and RNAs encoded by thenucleic acid molecules of the invention, and to polypeptides encoded bythese nucleic acid molecules or produced by the methods of theinvention, which may be fusion proteins. The invention also relates toantibodies that bind to one or more polypeptides of the invention orepitopes thereof, which may be monoclonal or polyclonal antibodies. Theinvention also relates to the use of these nucleic acid molecules,vectors, polypeptides and antibodies in methods for recombinationalcloning of nucleic acids, in vitro and in vivo, to provide chimeric DNAmolecules that have particular characteristics and/or DNA segments. Moreparticularly, the antibodies of the invention may be used to identifyand/or purify proteins or fusion proteins encoded by the nucleic acidmolecules or vectors of the invention, or to identify and/or purify thenucleic acid molecules of the invention.

2. Related Art

Site-Specific Recombinases.

Site-specific recombinases are proteins that are present in manyorganisms (e.g. viruses and bacteria) and have been characterized tohave both endonuclease and ligase properties. These recombinases (alongwith associated proteins in some cases) recognize specific sequences ofbases in DNA and exchange the DNA segments flanking those segments. Therecombinases and associated proteins are collectively referred to as“recombination proteins” (see, e.g., Landy, A., Current Opinion inBiotechnology 3:699-707 (1993)).

Numerous recombination systems from various organisms have beendescribed. See, e.g., Hoess et al., Nucleic Acids Research 14(6):2287(1986); Abremski et al., J. Biol. Chem. 261(1): 391 (1986); Campbell, J.Bacteriol. 174(23):7495 (1992); Qian et al., J. Biol. Chem. 267(11):7794(1992); Araki et al., J. Mol. Biol. 225(1):25 (1992); Maeser andKahnmann Mol. Gen. Genet. 230:170-176) (1991); Esposito et al., Nucl.Acids Res. 25(18):3605 (1997).

Many of these belong to the integrase family of recombinases (Argos etal. EMBO J. 5:433-440 (1986); Voziyanov et al., Nucl. Acids Res. 27:930(1999)). Perhaps the best studied of these are the Integrase/att systemfrom bacteriophage λ (Landy, A. Current Opinions in Genetics and Devel.3:699-707 (1993)), the Cre/loxP system from bacteriophage P1 (Hoess andAbremski (1990) In Nucleic Acids and Molecular Biology, vol. 4. Eds.:Eckstein and Lilley, Berlin-Heidelberg: Springer-Verlag; pp. 90-109),and the FLP/FRT system from the Saccharomyces cerevisiae 2μ circleplasmid (Broach et al. Cell 29:227-234 (1982)).

Backman (U.S. Pat. No. 4,673,640) discloses the in vivo use of λrecombinase to recombine a protein producing DNA segment by enzymaticsite-specific recombination using wild-type recombination sites attB andattP.

Hasan and Szybalski (Gene 56:145-151 (1987)) discloses the use of λ Intrecombinase in vivo for intramolecular recombination between wild typeattP and attB sites which flank a promoter. Because the orientations ofthese sites are inverted relative to each other, this causes anirreversible flipping of the promoter region relative to the gene ofinterest.

Palazzolo et al. Gene 88:25-36 (1990), discloses phage lambda vectorshaving bacteriophage λ arms that contain restriction sites positionedoutside a cloned DNA sequence and between wild-type loxP sites.Infection of E. coli cells that express the Cre recombinase with thesephage vectors results in recombination between the loxP sites and the invivo excision of the plasmid replicon, including the cloned cDNA.

Pósfai et al. (Nucl. Acids Res. 22:2392-2398 (1994)) discloses a methodfor inserting into genomic DNA partial expression vectors having aselectable marker, flanked by two wild-type FRT recognition sequences.FLP site-specific recombinase as present in the cells is used tointegrate the vectors into the genome at predetermined sites. Underconditions where the replicon is functional, this cloned genomic DNA canbe amplified.

Bebee et al. (U.S. Pat. No. 5,434,066) discloses the use ofsite-specific recombinases such as Cre for DNA containing two loxP sitesfor in vivo recombination between the sites.

Boyd (Nucl. Acids Res. 21:817-821 (1993)) discloses a method tofacilitate the cloning of blunt-ended DNA using conditions thatencourage intermolecular ligation to a dephosphorylated vector thatcontains a wild-type loxP site acted upon by a Cre site-specificrecombinase present in E. coli host cells.

Waterhouse et al. (WO 93/19172 and Nucleic Acids Res. 21 (9):2265(1993)) disclose an in vivo method where light and heavy chains of aparticular antibody were cloned in different phage vectors between loxPand loxP 511 sites and used to transfect new E. coli cells. Cre, actingin the host cells on the two parental molecules (one plasmid, onephage), produced four products in equilibrium: two differentcointegrates (produced by recombination at either loxP or loxP 511sites), and two daughter molecules, one of which was the desiredproduct.

Schlake & Bode (Biochemistry 33:12746-12751 (1994)) discloses an in vivomethod to exchange expression cassettes at defined chromosomallocations, each flanked by a wild type and a spacer-mutated FRTrecombination site. A double-reciprocal crossover was mediated incultured mammalian cells by using this FLP/FRT system for site-specificrecombination.

Hartley et al. (U.S. Pat. No. 5,888,732) disclose compositions andmethods for recombinational exchange of nucleic acid segments andmolecules, including for use in recombinational cloning of a variety ofnucleic acid molecules in vitro and in vivo, using a combination ofwildtype and mutated recombination sites and recombination proteins.

Transposases.

The family of enzymes, the transposases, has also been used to transfergenetic information between replicons. Transposons are structurallyvariable, being described as simple or compound, but typically encodethe recombinase gene flanked by DNA sequences organized in invertedorientations. Integration of transposons can be random or highlyspecific. Representatives such as Tn7, which are highly site-specific,have been applied to the in vivo movement of DNA segments betweenreplicons (Lucklow et al., J. Virol. 67:4566-4579 (1993)).

Devine and Boeke Nucl. Acids Res. 22:3765-3772 (1994), discloses theconstruction of artificial transposons for the insertion of DNAsegments, in vitro, into recipient DNA molecules. The system makes useof the integrase of yeast TY1 virus-like particles. The DNA segment ofinterest is cloned, using standard methods, between the ends of thetransposon-like element TY1. In the presence of the TY1 integrase, theresulting element integrates randomly into a second target DNA molecule.

Recombination Sites.

Also key to the integration/recombination reactions mediated by theabove-noted recombination proteins and/or transposases are recognitionsequences, often termed “recombination sites,” on the DNA moleculesparticipating in the integration/recombination reactions. Theserecombination sites are discrete sections or segments of DNA on theparticipating nucleic acid molecules that are recognized and bound bythe recombination proteins during the initial stages of integration orrecombination. For example, the recombination site for Cre recombinaseis loxP which is a 34 base pair sequence comprised of two 13 base pairinverted repeats (serving as the recombinase binding sites) flanking an8 base pair core sequence. See FIG. 1 of Sauer, B., Curr. Opin. Biotech.5:521-527 (1994). Other examples of recognition sequences include theattB, attP, attL, and attR sequences which are recognized by therecombination protein λ Int. attB is an approximately 25 base pairsequence containing two 9 base pair core-type Int binding sites and a 7base pair overlap region, while attP is an approximately 240 base pairsequence containing core-type Int binding sites and arm-type Int bindingsites as well as sites for auxiliary proteins integration host factor(IHF), FIS and excisionase (Xis). See Landy, Curr. Opin. Biotech.3:699-707 (1993); see also U.S. Pat. No. 5,888,732, which isincorporated by reference herein.

DNA Cloning.

The cloning of DNA segments currently occurs as a daily routine in manyresearch labs and as a prerequisite step in many genetic analyses. Thepurpose of these clonings is various, however, two general purposes canbe considered: (1) the initial cloning of DNA from large DNA or RNAsegments (chromosomes, YACs, PCR fragments, mRNA, etc.), done in arelative handful of known vectors such as pUC, pGem, pBlueScript, and(2) the subcloning of these DNA segments into specialized vectors forfunctional analysis. A great deal of time and effort is expended both inthe transfer of DNA segments from the initial cloning vectors to themore specialized vectors. This transfer is called subcloning.

The basic methods for cloning have been known for many years and havechanged little during that time. A typical cloning protocol is asfollows:

-   -   (1) digest the DNA of interest with one or two restriction        enzymes;    -   (2) gel purify the DNA segment of interest when known;    -   (3) prepare the vector by cutting with appropriate restriction        enzymes, treating with alkaline phosphatase, gel purify etc., as        appropriate;    -   (4) ligate the DNA segment to the vector, with appropriate        controls to eliminate background of uncut and self-ligated        vector;    -   (5) introduce the resulting vector into an E. coli host cell;    -   (6) pick selected colonies and grow small cultures overnight;    -   (7) make DNA minipreps; and    -   (8) analyze the isolated plasmid on agarose gels (often after        diagnostic restriction enzyme digestions) or by PCR.

The specialized vectors used for subcloning DNA segments arefunctionally diverse. These include but are not limited to: vectors forexpressing nucleic acid molecules in various organisms; for regulatingnucleic acid molecule expression; for providing tags to aid in proteinpurification or to allow tracking of proteins in cells; for modifyingthe cloned DNA segment (e.g., generating deletions); for the synthesisof probes (e.g., riboprobes); for the preparation of templates for DNAsequencing; for the identification of protein coding regions; for thefusion of various protein-coding regions; to provide large amounts ofthe DNA of interest, etc. It is common that a particular investigationwill involve subcloning the DNA segment of interest into severaldifferent specialized vectors.

As known in the art, simple subclonings can be done in one day (e.g.,the DNA segment is not large and the restriction sites are compatiblewith those of the subcloning vector). However, many other subcloningscan take several weeks, especially those involving unknown sequences,long fragments, toxic genes, unsuitable placement of restriction sites,high backgrounds, impure enzymes, etc. Subcloning DNA fragments is thusoften viewed as a chore to be done as few times as possible.

Several methods for facilitating the cloning of DNA segments have beendescribed, e.g., as in the following references.

Ferguson, J., et al. Gene 16:191 (1981), discloses a family of vectorsfor subcloning fragments of yeast DNA. The vectors encode kanamycinresistance. Clones of longer yeast DNA segments can be partiallydigested and ligated into the subcloning vectors. If the originalcloning vector conveys resistance to ampicillin, no purification isnecessary prior to transformation, since the selection will be forkanamycin.

Hashimoto-Gotoh, T., et al. Gene 41:125 (1986), discloses a subcloningvector with unique cloning sites within a streptomycin sensitivity gene;in a streptomycin-resistant host, only plasmids with inserts ordeletions in the dominant sensitivity gene will survive streptomycinselection.

Accordingly, traditional subcloning methods, using restriction enzymesand ligase, are time consuming and relatively unreliable. Considerablelabor is expended, and if two or more days later the desired subclonecan not be found among the candidate plasmids, the entire process mustthen be repeated with alternative conditions attempted. Although sitespecific recombinases have been used to recombine DNA in vivo, thesuccessful use of such enzymes in vitro was expected to suffer fromseveral problems. For example, the site specificities and efficiencieswere expected to differ in vitro; topologically linked products wereexpected; and the topology of the DNA substrates and recombinationproteins was expected to differ significantly in vitro (see, e.g., Adamset al, J. Mol. Biol. 226:661-73 (1992)). Reactions that could go on formany hours in vivo were expected to occur in significantly less time invitro before the enzymes became inactive. In addition, the stabilitiesof the recombination enzymes after incubation for extended periods oftime in in vitro reactions was unknown, as were the effects of thetopologies (i.e., linear, coiled, supercoiled, etc.) of the nucleic acidmolecules involved in the reaction. Multiple DNA recombination productswere expected in the biological host used, resulting in unsatisfactoryreliability, specificity or efficiency of subcloning. Thus, in vitrorecombination reactions were not expected to be sufficiently efficientto yield the desired levels of product.

Accordingly, there is a long felt need to provide an alternativesubcloning system that provides advantages over the known use ofrestriction enzymes and ligases.

SUMMARY OF THE INVENTION

The present invention relates to nucleic acid molecules encoding one ormore recombination sites or one or more partial recombination sites,particularly attB, attP, attL, and attR, and fragments, mutants,variants and derivatives thereof. The invention also relates to suchnucleic acid molecules comprising one or more of the recombination sitenucleotide sequences or portions thereof and one or more additionalphysical or functional nucleotide sequences, such as those encoding oneor more multiple cloning sites, one or more transcription terminationsites, one or more transcriptional regulatory sequences (e.g., one ormore promoters, enhancers, or repressors), one or more translationalsignal sequences, one or more nucleotide sequences encoding a fusionpartner protein or peptide (e.g., GST, His₆ or thioredoxin), one or moreselection markers or modules, one or more nucleotide sequences encodinglocalization signals such as nuclear localization signals or secretionsignals, one or more origins of replication, one or more proteasecleavage sites, one or more desired proteins or peptides encoded by agene or a portion of a gene, and one or more 5′ or 3′ polynucleotidetails (particularly a poly-G tail). The invention also relates to suchnucleic acid molecules wherein the one or more recombination sitenucleotide sequences is operably linked to the one or more additionalphysical or functional nucleotide sequences.

The invention also relates to primer nucleic acid molecules comprisingthe recombination site nucleotide sequences of the invention (orportions thereof), and to such primer nucleic acid molecules linked toone or more target-specific (e.g., one or more gene-specific) primernucleic acid sequences. Such primers may also comprise sequencescomplementary or homologous to DNA or RNA sequences to be amplified,e.g., by PCR, RT-PCR, etc. Such primers may also comprise sequences orportions of sequences useful in the expression of protein genes(ribosome binding sites, localization signals, protease cleavage sites,repressor binding sites, promoters, transcription stops, stop codons,etc.). Said primers may also comprise sequences or portions of sequencesuseful in the manipulation of DNA molecules (restriction sites,transposition sites, sequencing primers, etc.). The primers of theinvention may be used in nucleic acid synthesis and preferably are usedfor amplification (e.g., PCR) of nucleic acid molecules. When theprimers of the invention include target- or gene-specific sequences (anysequence contained within the target to be synthesized or amplifiedincluding translation signals, gene sequences, stop codons,transcriptional signals (e.g., promoters) and the like), amplificationor synthesis of target sequences or genes may be accomplished. Thus, theinvention relates to synthesis of a nucleic acid molecules comprisingmixing one or more primers of the invention with a nucleic acidtemplate, and incubating said mixture under conditions sufficient tomake a first nucleic acid molecule complementary to all or a portion ofsaid template. Thus, the invention relates specifically to a method ofsynthesizing a nucleic acid molecule comprising:

-   -   (a) mixing a nucleic acid template with a polypeptide having        polymerase activity and one or more primers comprising one or        more recombination sites or portions thereof; and    -   (b) incubating said mixture under conditions sufficient to        synthesize a first nucleic acid molecule complementary to all or        a portion of said template and which preferably comprises one or        more recombination sites or portions thereof.        Such method of the invention may further comprise incubating        said first synthesized nucleic acid molecule under conditions        sufficient to synthesize a second nucleic acid molecule        complementary to all or a portion of said first nucleic acid        molecule. Such synthesis may provide for a first nucleic acid        molecule having a recombination site or portion thereof at one        or both of its termini.

In a preferred aspect, for the synthesis of the nucleic acid molecules,at least two primers are used wherein each primer comprises a homologoussequence at its terminus and/or within internal sequences of each primer(which may have a homology length of about 2 to about 500 bases,preferably about 3 to about 100 bases, about 4 to about 50 bases, about5 to about 25 bases and most preferably about 6 to about 18 baseoverlap). In a preferred aspect, the first such primer comprises atleast one target-specific sequence and at least one recombination siteor portion thereof while the second primer comprises at least onerecombination site or portion thereof. Preferably, the homologousregions between the first and second primers comprise at least a portionof the recombination site. In another aspect, the homologous regionsbetween the first and second primers may comprise one or more additionalsequences, e.g., expression signals, translational start motifs, orother sequences adding functionality to the desired nucleic acidsequence upon amplification. In practice, two pairs of primers primesynthesis or amplification of a nucleic acid molecule. In a preferredaspect, all or at least a portion of the synthesized or amplifiednucleic acid molecule will be homologous to all or a portion of thetemplate and further comprises a recombination site or a portion thereofat least one terminus and preferably both termini of the synthesized oramplified molecule. Such synthesized or amplified nucleic acid moleculemay be double stranded or single stranded and may be used in therecombinational cloning methods of the invention. The homologous primersof the invention provide a substantial advantage in that one set of theprimers may be standardized for any synthesis or amplification reaction.That is, the primers providing the recombination site sequences (withoutthe target specific sequences) can be pre-made and readily available foruse. This in practice allows the use of shorter custom made primers thatcontain the target specific sequence needed to synthesize or amplify thedesired nucleic acid molecule. Thus, this provides reduced time and costin preparing target specific primers (e.g., shorter primers containingthe target specific sequences can be prepared and used in synthesisreactions). The standardized primers, on the other hand, may be producedin mass to reduce cost and can be readily provided (e.g., in kits or asa product) to facilitate synthesis of the desired nucleic acidmolecules.

Thus, in one preferred aspect, the invention relates to a method ofsynthesizing or amplifying one or more nucleic acid moleculescomprising:

-   -   (a) mixing one or more nucleic acid templates with at least one        polypeptide having polymerase or reverse transcriptase activity        and at least a first primer comprising a template specific        sequence (complementary to or capable of hybridizing to said        templates) and at least a second primer comprising all or a        portion of a recombination site wherein said at least a portion        of said second primer is homologous to or complementary to at        least a portion of said first primer; and    -   (b) incubating said mixture under conditions sufficient to        synthesize or amplify one or more nucleic acid molecules        complementary to all or a portion of said templates and        comprising one or more recombination sites or portions thereof        at one and preferably both termini of said molecules.

More specifically, the invention relates to a method of synthesizing oramplifying one or more nucleic acid molecules comprising:

-   -   (a) mixing one or more nucleic acid templates with at least one        polypeptide having polymerase or reverse transcriptase activity        and at least a first primer comprising a template specific        sequence (complementary to or capable of hybridizing to said        templates) and at least a portion of, a recombination site, and        at least a second primer comprising all or a portion of a        recombination site wherein said at least a portion of said        recombination site on said second primer is complementary to or        homologous to at least a portion of said recombination site on        said first primer; and    -   (b) incubating said mixture under conditions sufficient to        synthesize or amplify one or more nucleic acid molecules        complementary to all or a portion of said templates and        comprising one or more recombination sites or portions thereof        at one and preferably both termini of said molecules.

In a more preferred aspect, the invention relates to a method ofamplifying or synthesizing one or more nucleic acid moleculescomprising:

-   -   (a) mixing one or more nucleic acid templates with at least one        polypeptide having polymerase or reverse transcriptase activity        and one or more first primers comprising at least a portion of a        recombination site and a template specific sequence        (complementary to or capable of hybridizing to said template);    -   (b) incubating said mixture under conditions sufficient to        synthesize or amplify one or more first nucleic acid molecules        complementary to all or a portion of said templates wherein said        molecules comprise at least a portion of a recombination site at        one and preferably both termini of said molecules;    -   (c) mixing said molecules with one or more second primers        comprising one or more recombination sites, wherein said        recombination sites of said second primers are homologous to or        complementary to at least a portion of said recombination sites        on said first nucleic acid molecules; and    -   (d) incubating said mixture under conditions sufficient to        synthesize or amplify one or more second nucleic acid molecules        complementary to all or a portion of said first nucleic acid        molecules and which comprise one or more recombination sites at        one and preferably both termini of said molecules.

The invention also relates to vectors comprising the nucleic acidmolecules of the invention, to host cells comprising the vectors ornucleic acid molecules of the invention, to methods of producingpolypeptides encoded by the nucleic acid molecules of the invention, andto polypeptides encoded by these nucleic acid molecules or produced bythe methods of the invention, which may be fusion proteins. Theinvention also relates to antibodies that bind to one or morepolypeptides of the invention or epitopes thereof, which may bemonoclonal or polyclonal antibodies. The invention also relates to theuse of these nucleic acid molecules, primers, vectors, polypeptides andantibodies in methods for recombinational cloning of nucleic acids, invitro and in vivo, to provide chimeric DNA molecules that haveparticular characteristics and/or DNA segments.

The antibodies of the invention may have particular use to identifyand/or purify peptides or proteins (including fusion proteins producedby the invention), and to identify and/or purify the nucleic acidmolecules of the invention or portions thereof.

The methods for in vitro or in vivo recombinational cloning of nucleicacid molecule generally relate to recombination between at least a firstnucleic acid molecule having at least one recombination site and asecond nucleic acid molecule having at least one recombination site toprovide a chimeric nucleic acid molecule. In one aspect, the methodsrelate to recombination between and first vector having at least onerecombination site and a second vector having at least one recombinationsite to provide a chimeric vector. In another aspect, a nucleic acidmolecule having at least one recombination site is combined with avector having at least one recombination site to provide a chimericvector. In a most preferred aspect, the nucleic acid molecules orvectors used in recombination comprise two or more recombination sites.In a more specific embodiment of the invention, the recombinationmethods relate to a Destination Reaction (also referred to herein as an“LR reaction”) in which recombination occurs between an Entry clone anda Destination Vector. Such a reaction transfers the nucleic acidmolecule of interest from the Entry Clone into the Destination Vector tocreate an Expression Clone. The methods of the invention alsospecifically relate to an Entry or Gateward reaction (also referred toherein as a “BP reaction”) in which an Expression Clone is recombinedwith a Donor vector to produce an Entry clone. In other aspects, theinvention relates to methods to prepare Entry clones by combining anEntry vector with at least one nucleic acid molecule (e.g., gene orportion of a gene). The invention also relates to conversion of adesired vector into a Destination Vector by including one or more(preferably at least two) recombination sites in the vector of interest.In a more preferred aspect, a nucleic acid molecule (e.g., a cassette)having at least two recombination sites flanking a selectable marker(e.g., a toxic gene or a genetic element preventing the survival of ahost cell containing that gene or element, and/or preventingreplication, partition or heritability of a nucleic acid molecule (e.g.,a vector or plasmid) comprising that gene or element) is added to thevector to make a Destination Vector of the invention.

Preferred vectors for use in the invention include prokaryotic vectors,eukaryotic vectors, or vectors which may shuttle between variousprokaryotic and/or eukaryotic systems (e.g. shuttle vectors). Preferredprokaryotic vectors for use in the invention include but are not limitedto vectors which may propagate and/or replicate in gram negative and/orgram positive bacteria, including bacteria of the genera Escherichia,Salmonella, Proteus, Clostridium, Klebsiella, Bacillus, Streptomyces,and Pseudomonas and preferably in the species E. coli. Eukaryoticvectors for use in the invention include vectors which propagate and/orreplicate and yeast cells, plant cells, mammalian cells, (particularlyhuman and mouse), fungal cells, insect cells, nematode cells, fish cellsand the like. Particular vectors of interest include but are not limitedto cloning vectors, sequencing vectors, expression vectors, fusionvectors, two-hybrid vectors, gene therapy vectors, phage displayvectors, gene-targeting vectors, PACs, BACs, YACs, MACs, and reversetwo-hybrid vectors. Such vectors may be used in prokaryotic and/oreukaryotic systems depending on the particular vector.

In another aspect, the invention relates to kits which may be used incarrying out the methods of the invention, and more specifically relatesto cloning or subcloning kits and kits for carrying out the LR Reaction(e.g., making an Expression Clone), for carrying out the BP Reaction(e.g., making an Entry Clone), and for making Entry Clone andDestination Vector molecules of the invention. Such kits may comprise acarrier or receptacle being compartmentalized to receive and holdtherein any number of containers. Such containers may contain any numberof components for carrying out the methods of the invention orcombinations of such components. In particular, a kit of the inventionmay comprise one or more components (or combinations thereof) selectedfrom the group consisting of one or more recombination proteins orauxiliary factors or combinations thereof, one or more compositionscomprising one or more recombination proteins or auxiliary factors orcombinations thereof (for example, GATEWAY™ LR Clonase™ Enzyme Mix orGATEWAY™ BP Clonase™ Enzyme Mix), one or more reaction buffers, one ormore nucleotides, one or more primers of the invention, one or morerestriction enzymes, one or more ligases, one or more polypeptideshaving polymerase activity (e.g., one or more reverse transcriptases orDNA polymerases), one or more proteinases (e.g., proteinase K or otherproteinases), one or more Destination Vector molecules, one or moreEntry Clone molecules, one or more host cells (e.g. competent cells,such as E. coli cells, yeast cells, animal cells (including mammaliancells, insect cells, nematode cells, avian cells, fish cells, etc.),plant cells, and most particularly E. coli DB3.1 host cells, such as E.coli LIBRARY EFFICIENCY® DB3.1™ Competent Cells), instructions for usingthe kits of the invention (e.g., to carry out the methods of theinvention), and the like. In related aspects, the kits of the inventionmay comprise one or more nucleic acid molecules encoding one or morerecombination sites or portions thereof, particularly one or morenucleic acid molecules comprising a nucleotide sequence encoding the oneor more recombination sites or portions thereof of the invention.Preferably, such nucleic acid molecules comprise at least tworecombination sites which flank a selectable marker (e.g., a toxic geneand/or antibiotic resistance gene). In a preferred aspect, such nucleicacid molecules are in the form of a cassette (e.g., a linear nucleicacid molecule comprising one or more and preferably two or morerecombination sites or portions thereof).

Kits for inserting or adding recombination sites to nucleic acidmolecules of interest may comprise one or more nucleases (preferablyrestriction endonucleases), one or more ligases, one or moretopoisomerases, one or more polymerases, and one or more nucleic acidmolecules or adapters comprising one or more recombination sites. Kitsfor integrating recombination sites into one or more nucleic acidmolecules of interest may comprise one or more components (orcombinations thereof) selected from the group consisting of one or moreintegration sequences comprising one or more recombination sites. Suchintegration sequences may comprise one or more transposons, integratingviruses, homologous recombination sequences, RNA molecules, one or morehost cells and the like.

Kits for making the Entry Clone molecules of the invention may compriseany or a number of components and the composition of such kits may varydepending on the specific method involved. Such methods may involveinserting the nucleic acid molecules of interest into an Entry or DonorVector by the recombinational cloning methods of the invention, or usingconventional molecular biology techniques (e.g., restriction enzymedigestion and ligation). In a preferred aspect, the Entry Clone is madeusing nucleic acid amplification or synthesis products. Kits forsynthesizing Entry Clone molecules from amplification or synthesisproducts may comprise one or more components (or combinations thereof)selected from the group consisting of one or more Donor Vectors (e.g.,one or more attP vectors including, but not limited to, pDONR201 (FIG.49), pDONR202 (FIG. 50), pDONR203 (FIG. 51), pDONR204 (FIG. 52),pDONR205 (FIG. 53), pDONR206 (FIG. 53), and the like), one or morepolypeptides having polymerase activity (preferably DNA polymerases andmost preferably thermostable DNA polymerases), one or more proteinases,one or more reaction buffers, one or more nucleotides, one or moreprimers comprising one or more recombination sites or portions thereof,and instructions for making one or more Entry Clones.

Kits for making the Destination vectors of the invention may compriseany number of components and the compositions of such kits may varydepending on the specific method involved. Such methods may include therecombination methods of the invention or conventional molecular biologytechniques (e.g., restriction endonuclease digestion and ligation). In apreferred aspect, the Destination vector is made by inserting a nucleicacid molecule comprising at least one recombination site (or portionthereof) of the invention (preferably a nucleic acid molecule comprisingat least two recombination sites or portions thereof flanking aselectable marker) into a desired vector to convert the desired vectorinto a Destination vector of the invention. Such kits may comprise atleast one component (or combinations thereof) selected from the groupconsisting of one or more restriction endonucleases, one or moreligases, one or more polymerases, one or more nucleotides, reactionbuffers, one or more nucleic acid molecules comprising at least onerecombination site or portion thereof (preferably at least one nucleicacid molecule comprising at least two recombination sites flanking atleast one selectable marker, such as a cassette comprising at least oneselectable marker such as antibiotic resistance genes and/or toxicgenes), and instructions for making such Destination vectors.

The invention also relates to kits for using the antibodies of theinvention in identification and/or isolation of peptides and proteins(which may be fusion proteins) produced by the nucleic acid molecules ofthe invention, and for identification and/or isolation of the nucleicacid molecules of the invention or portions thereof. Such kits maycomprise one or more components (or combination thereof) selected fromthe group consisting of one or more antibodies of the invention, one ormore detectable labels, one or more solid supports and the like.

Other preferred embodiments of the present invention will be apparent toone of ordinary skill in light of what is known in the art, in light ofthe following drawings and description of the invention, and in light ofthe claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts one general method of the present invention, wherein thestarting (parent) DNA molecules can be circular or linear. The goal isto exchange the new subcloning vector D for the original cloning vectorB. It is desirable in one embodiment to select for AD and against allthe other molecules, including the Cointegrate. The square and circleare sites of recombination: e.g., lox (such as loxP) sites, att sites,etc. For example, segment D can contain expression signals, proteinfusion domains, new drug markers, new origins of replication, orspecialized functions for mapping or sequencing DNA. It should be notedthat the cointegrate molecule contains Segment D (Destination vector)adjacent to segment A (Insert), thereby juxtaposing functional elementsin D with the insert in A. Such molecules can be used directly in vitro(e.g., if a promoter is positioned adjacent to a gene-for in vitrotranscription/translation) or in vivo (following isolation in a cellcapable of propagating ccdB-containing vectors) by selecting for theselection markers in Segments B+D. As one skilled in the art willrecognize, this single step method has utility in certain envisionedapplications of the invention.

FIG. 2 is a more detailed depiction of the recombinational cloningsystem of the invention, referred to herein as the “GATEWAY™ CloningSystem.” This figure depicts the production of Expression Clones via a“Destination Reaction,” which may also be referred to herein as an “LRReaction.” A kan^(r) vector (referred to herein as an “Entry clone”)containing a DNA molecule of interest (e.g., a gene) localized betweenan attL1 site and an attL2 site is reacted with an amp^(r) vector(referred to herein as a “Destination Vector”) containing a toxic or“death” gene localized between an attR1 site and an attR2 site, in thepresence of GATEWAY™ LR Clonase™ Enzyme Mix (a mixture of Int, IHF andXis). After incubation at 25° C. for about 60 minutes, the reactionyields an amp^(r) Expression Clone containing the DNA molecule ofinterest localized between an attB1 site and an attB2 site, and akan^(r) byproduct molecule, as well as intermediates. The reactionmixture may then be transformed into host cells (e.g., E. coli) andclones containing the nucleic acid molecule of interest may be selectedby plating the cells onto ampicillin-containing media and pickingamp^(r) colonies.

FIG. 3 is a schematic depiction of the cloning of a nucleic acidmolecule from an Entry clone into multiple types of Destination vectors,to produce a variety of Expression Clones. Recombination between a givenEntry clone and different types of Destination vectors (not shown), viathe LR Reaction depicted in FIG. 2, produces multiple differentExpression Clones for use in a variety of applications and host celltypes.

FIG. 4 is a detailed depiction of the production of Entry Clones via a“BP reaction,” also referred to herein as an “Entry Reaction” or a“Gateward Reaction.” In the example shown in this figure, an amp^(r)expression vector containing a DNA molecule of interest (e.g., a gene)localized between an attB1 site and an attB2 site is reacted with akan^(r) Donor vector (e.g., an attP vector; here, GATEWAY™ pDONR201 (seeFIG. 49A-C)) containing a toxic or “death” gene localized between anattP1 site and an attP2 site, in the presence of GATEWAY™ BP Clonase™Enzyme Mix (a mixture of Int and IHF). After incubation at 25° C. forabout 60 minutes, the reaction yields a kan^(r) Entry clone containingthe DNA molecule of interest localized between an attL1 site and anattL2 site, and an amp^(r) by-product molecule. The Entry clone may thenbe transformed into host cells (e.g., E. coli) and clones containing theEntry clone (and therefore the nucleic acid molecule of interest) may beselected by plating the cells onto kanamycin-containing media andpicking kan^(r) colonies. Although this figure shows an example of useof a kan^(r) Donor vector, it is also possible to use Donor vectorscontaining other selection markers, such as the gentamycin resistance ortetracycline resistance markers, as discussed herein.

FIG. 5 is a more detailed schematic depiction of the LR (“Destination”)reaction (FIG. 5A) and the BP (“Entry” or “Gateward”) reaction (FIG. 5B)of the GATEWAY™ Cloning System, showing the reactants, products andbyproducts of each reaction.

FIG. 6 shows the sequences of the attB1 (SEQ ID NO:1) and attB2 (SEQ IDNO:2) sites flanking a gene of interest after subcloning into aDestination Vector to create an Expression Clone.

FIG. 7 is a schematic depiction of four ways to make Entry Clones usingthe compositions and methods of the invention: 1. using restrictionenzymes and ligase; 2. starting with a cDNA library prepared in an attLEntry Vector; 3. using an Expression Clone from a library prepared in anattB Expression Vector via the BxP reaction; and 4. recombinationalcloning of PCR fragments with terminal attB sites, via the BxP reaction.Approaches 3 and 4 rely on recombination with a Donor vector (here, anattP vector such as pDONR201 (see FIG. 49A-C), pDONR202 (see FIG.50A-C), pDONR203 (see FIG. 51A-C), pDONR204 (see FIG. 52A-C), pDONR205(see FIG. 53A-C), or pDONR206 (see FIG. 54A-C), for example) thatprovides an Entry Clone carrying a selection marker such as kan^(r),gen^(r), tet^(r), or the like.

FIG. 8 is a schematic depiction of cloning of a PCR product by a BxP(Entry or Gateward) reaction. A PCR product with 25 bp terminal attBsites (plus four Gs) is shown as a substrate for the BxP reaction.Recombination between the attB-PCR product of a gene and a Donor vector(which donates an Entry Vector that carries kan^(r)) results in an EntryClone of the PCR product.

FIG. 9 is a listing of the nucleotide sequences of the recombinationsites designated herein as attB1, attB2, attP1, attP2, attL1, attL2,attR1 and attR2 (SEQ ID NOs:1-8, respectively). Sequences are writtenconventionally, from 5′ to 3′.

FIGS. 10-20: The plasmid backbone for all the Entry Vectors depictedherein is the same, and is shown in FIG. 10A for the Entry VectorpENTR1A. For other Entry Vectors shown in FIGS. 11-20, only thesequences shown in Figure “A” for each figure set (i.e., FIG. 11A, FIG.12A, etc.) are different (within the attL1-attL2 cassettes) from thoseshown in FIG. 10—the plasmid backbone is identical.

FIG. 10 is a schematic depiction of the physical map and cloning sites(FIG. 10A), and the nucleotide sequence (FIG. 10B) (SEQ ID NO:118), ofthe Entry Vector pENTR1A.

FIG. 11 is a schematic depiction of the cloning sites (FIG. 11A) and thenucleotide sequence (FIG. 11B) (SEQ ID NO:119) of the Entry VectorpENTR2B.

FIG. 12 is a schematic depiction of the cloning sites (FIG. 12A) and thenucleotide sequence (FIG. 12B) (SEQ ID NO:120) of the Entry VectorpENTR3C.

FIG. 13 is a schematic depiction of the cloning sites (FIG. 13A) and thenucleotide sequence (FIG. 13B) (SEQ ID NO:121) of the Entry VectorpENTR4.

FIG. 14 is a schematic depiction of the cloning sites (FIG. 14A) and thenucleotide sequence (FIG. 14B) (SEQ ID NO:122) of the Entry VectorpENTR5.

FIG. 15 is a schematic depiction of the cloning sites (FIG. 15A) and thenucleotide sequence (FIG. 15B) (SEQ ID NO:123) of the Entry VectorpENTR6.

FIG. 16 is a schematic depiction of the cloning sites (FIG. 16A) and thenucleotide sequence (FIG. 16B) (SEQ ID NO:124) of the Entry VectorpENTR7.

FIG. 17 is a schematic depiction of the cloning sites (FIG. 17A) and thenucleotide sequence (FIG. 17B) (SEQ ID NO:125) of the Entry VectorpENTR8.

FIG. 18 is a schematic depiction of the cloning sites (FIG. 18A) and thenucleotide sequence (FIG. 18B) (SEQ ID NO:126) of the Entry VectorpENTR9.

FIG. 19 is a schematic depiction of the cloning sites (FIG. 19A) and thenucleotide sequence (FIG. 19B) (SEQ ID NO:127) of the Entry VectorpENTR10.

FIG. 20 is a schematic depiction of the cloning sites (FIG. 20A) and thenucleotide sequence (FIG. 20B) (SEQ ID NO:128) of the Entry VectorpENTR11.

FIG. 21 is a schematic depiction of the physical map and the Trcexpression cassette (FIG. 21A) showing the promoter sequences at −35 andat −10 from the initiation codon, and the nucleotide sequence (FIG.21B-D) (SEQ ID NO:129), of Destination Vector pDEST1. This vector mayalso be referred to as pTrc-DEST1.

FIG. 22 is a schematic depiction of the physical map and the His6expression cassette (FIG. 22A) showing the promoter sequences at −35 andat −10 from the initiation codon, and the nucleotide sequence (FIG.22B-D) (SEQ ID NO:130), of Destination Vector pDEST2. This vector mayalso be referred to as pHis6-DEST2.

FIG. 23 is a schematic depiction of the physical map and the GSTexpression cassette (FIG. 23A) showing the promoter sequences at −35 andat −10 from the initiation codon, and the nucleotide sequence (FIG.23B-D) (SEQ ID NO:131), of Destination Vector pDEST3. This vector mayalso be referred to as pGST-DEST3.

FIG. 24 is a schematic depiction of the physical map and the His6-Trxexpression cassette (FIG. 24A) showing the promoter sequences at −35 andat −10 from the initiation codon and a TEV protease cleavage site, andthe nucleotide sequence (FIG. 24B-D) (SEQ ID NO:132), of DestinationVector pDEST4. This vector may also be referred to as pTrx-DEST4.

FIG. 25 is a schematic depiction of the attR1 and attR2 sites (FIG.25A), the physical map (FIG. 25B), and the nucleotide sequence (FIGS.25C-E) (SEQ ID NO:133), of Destination Vector pDEST5. This vector mayalso be referred to as pSPORT(+)-DEST5.

FIG. 26 is a schematic depiction of the attR1 and attR2 sites (FIG.26A), the physical map (FIG. 26B), and the nucleotide sequence (FIGS.26C-E) (SEQ ID NO:134), of Destination Vector pDEST6. This vector mayalso be referred to as pSPORT(−)-DEST6.

FIG. 27 is a schematic depiction of the attR1 site, CMV promoter, andthe physical map (FIG. 27A), and the nucleotide sequence (FIGS. 27B-D)(SEQ ID NO:135), of Destination Vector pDEST7. This vector may also bereferred to as pCMV-DEST7.

FIG. 28 is a schematic depiction of the attR1 site, baculoviruspolyhedrin promoter, and the physical map (FIG. 28A), and the nucleotidesequence (FIG. 28B-D) (SEQ ID NO:136), of Destination Vector pDEST8.This vector may also be referred to as pFastBac-DEST8.

FIG. 29 is a schematic depiction of the attR1 site, Semliki Forest Viruspromoter, and the physical map (FIG. 29A), and the nucleotide sequence(FIGS. 29B-G) (SEQ ID NO:137), of Destination Vector pDEST9. This vectormay also be referred to as pSFV-DEST9.

FIG. 30 is a schematic depiction of the attR1 site, baculoviruspolyhedrin promoter, His6 fusion domain, and the physical map (FIG.30A), and the nucleotide sequence (FIG. 30B-D) (SEQ ID NO:138), ofDestination Vector pDEST10. This vector may also be referred to aspFastBacHT-DEST10.

FIG. 31 is a schematic depiction of the attR1 cassette containing atetracycline-regulated CMV promoter and the physical map (FIG. 31A), andthe nucleotide sequence (FIGS. 31B-E) (SEQ ID NO:139), of DestinationVector pDEST11. This vector may also be referred to as pTet-DEST11.

FIG. 32 is a schematic depiction of the attR1 site, the start of themRNA of the CMV promoter, and the physical map (FIG. 32A), and thenucleotide sequence (FIGS. 32B-E) (SEQ ID NO:140), of Destination VectorpDEST12.2. This vector may also be referred to as pCMVneo-DEST12, aspCMV-DEST12, or as pDEST12.

FIG. 33 is a schematic depiction of the attR1 site, the λP_(L) promoter,and the physical map (FIG. 33A), and the nucleotide sequence (FIGS.33B-D) (SEQ ID NO:141), of Destination Vector pDEST13. This vector mayalso be referred to as pλP_(L)-DEST13.

FIG. 34 is a schematic depiction of the attR1 site, the T7 promoter, andthe physical map (FIG. 34A), and the nucleotide sequence (FIG. 34B-D)(SEQ ID NO:142), of Destination Vector pDEST14. This vector may also bereferred to as pPT7-DEST14.

FIG. 35 is a schematic depiction of the attR1 site, the T7 promoter, andthe N-terminal GST fusion sequence, and the physical map (FIG. 35A), andthe nucleotide sequence (FIG. 35B-D) (SEQ ID NO:143), of DestinationVector pDEST15. This vector may also be referred to as p17 GST-DEST15.

FIG. 36 is a schematic depiction of the attR1 site, the 17 promoter, andthe N-terminal thioredoxin fusion sequence, and the physical map (FIG.36A), and the nucleotide sequence (FIG. 36B-D) (SEQ ID NO:144), ofDestination Vector pDEST16. This vector may also be referred to as pT7Trx-DEST16.

FIG. 37 is a schematic depiction of the attR1 site, the 17 promoter, andthe N-terminal His6 fusion sequence, and the physical map (FIG. 37A),and the nucleotide sequence (FIG. 37B-D) (SEQ ID NO:145), of DestinationVector pDEST17. This vector may also be referred to as pT7 His-DEST17.

FIG. 38 is a schematic depiction of the attR1 site and the p10baculovirus promoter, and the physical map (FIG. 38A), and thenucleotide sequence (FIG. 38B-D) (SEQ ID NO:146), of Destination VectorpDEST18. This vector may also be referred to as pFBp10-DEST18.

FIG. 39 is a schematic depiction of the attR1 site, and the 39kbaculovirus promoter, and the physical map (FIG. 39A), and thenucleotide sequence (FIG. 39B-D) (SEQ ID NO:147), of Destination VectorpDEST19. This vector may also be referred to as pFB39k-DEST19.

FIG. 40 is a schematic depiction of the attR1 site, the polh baculoviruspromoter, and the N-terminal GST fusion sequence, and the physical map(FIG. 40A), and the nucleotide sequence (FIG. 40B-D) (SEQ ID NO:148), ofDestination Vector pDEST20. This vector may also be referred to as pFBGST-DEST20.

FIG. 41 is a schematic depiction of a 2-hybrid vector with a DNA-bindingdomain, the attR1 site, and the ADH promoter, and the physical map (FIG.41A), and the nucleotide sequence (FIGS. 41B-F) (SEQ ID NO:149), ofDestination Vector pDEST21. This vector may also be referred to as pDBLeu-DEST21.

FIG. 42 is a schematic depiction of a 2-hybrid vector with an activationdomain, the attR1 site, and the ADH promoter, and the physical map (FIG.42A), and the nucleotide sequence (FIGS. 42B-E) (SEQ ID NO:150), ofDestination Vector pDEST22. This vector may also be referred to aspPC86-DEST22.

FIG. 43 is a schematic depiction of the attR1 and attR2 sites, the T7promoter, and the C-terminal His6 fusion sequence, and the physical map(FIG. 43A), and the nucleotide sequence (FIG. 43B-D) (SEQ ID NO:151), ofDestination Vector pDEST23. This vector may also be referred to aspC-term-His6-DEST23.

FIG. 44 is a schematic depiction of the attR1 and attR2 sites, the T7promoter, and the C-terminal GST fusion sequence, and the physical map(FIG. 44A), and the nucleotide sequence (FIG. 44B-D) (SEQ ID NO:152), ofDestination Vector pDEST24. This vector may also be referred to aspC-term-GST-DEST24.

FIG. 45 is a schematic depiction of the attR1 and attR2 sites, the T7promoter, and the C-terminal thioredoxin fusion sequence, and thephysical map (FIG. 45A), and the nucleotide sequence (FIG. 45B-D) (SEQID NO:153), of Destination Vector pDEST25. This vector may also bereferred to as pC-term-Trx-DEST25.

FIG. 46 is a schematic depiction of the attR1 site, the CMV promoter,and an N-terminal His6 fusion sequence, and the physical map (FIG. 46A),and the nucleotide sequence (FIG. 46B-D) (SEQ ID NO:154), of DestinationVector pDEST26. This vector may also be referred to aspCMV-SPneo-His-DEST26.

FIG. 47 is a schematic depiction of the attR1 site, the CMV promoter,and an N-terminal GST fusion sequence, and the physical map (FIG. 47A),and the nucleotide sequence (FIGS. 47B-E) (SEQ ID NO:155), ofDestination Vector pDEST27. This vector may also be referred to aspCMV-Spneo-GST-DEST27.

FIG. 48 is a depiction of the physical map (FIG. 48A), the cloning sites(FIG. 48B), and the nucleotide sequence (FIG. 48C-D) (SEQ ID NO:156),for the attB cloning vector plasmid pEXP501. This vector may also bereferred to equivalently herein as pCMV.SPORT6, pCMVSPORT6, andpCMVSport6.

FIG. 49 is a depiction of the physical map (FIG. 49A), and thenucleotide sequence (FIG. 49B-C) (SEQ ID NO:157), for the Donor plasmidpDONR201 which donates a kanamycin-resistant vector in the BP Reaction.This vector may also be referred to as pAttPkanr Donor Plasmid, or aspAttPkan Donor Plasmid

FIG. 50 is a depiction of the physical map (FIG. 50A), and thenucleotide sequence (FIG. 50B-C) (SEQ ID NO:158), for the Donor plasmidpDONR202 which donates a kanamycin-resistant vector in the BP Reaction.

FIG. 51 is a depiction of the physical map (FIG. 51A), and thenucleotide sequence (FIG. 51B-C) (SEQ ID NO:159), for the Donor plasmidpDONR203 which donates a kanamycin-resistant vector in the BP Reaction.

FIG. 52 is a depiction of the physical map (FIG. 52A), and thenucleotide sequence (FIG. 52B-C) (SEQ ID NO:160), for the Donor plasmidpDONR204 which donates a kanamycin-resistant vector in the BP Reaction.

FIG. 53 is a depiction of the physical map (FIG. 53A), and thenucleotide sequence (FIG. 53B-C) (SEQ ID NO:161), for the Donor plasmidpDONR205 which donates a tetracycline-resistant vector in the BPReaction.

FIG. 54 is a depiction of the physical map (FIG. 54A), and thenucleotide sequence (FIG. 54B-C) (SEQ ID NO:162), for the Donor plasmidpDONR206 which donates a gentamycin-resistant vector in the BP Reaction.This vector may also be referred to as pENTR22 attP Donor Plasmid,pAttPGenr Donor Plasmid, or pAttPgent Donor Plasmid.

FIG. 55 depicts the attB1 site, and the physical map, of an Entry Clone(pENTR7) of CAT subcloned into the Destination Vector pDEST2 (FIGS.22A-D)

FIG. 56 depicts the DNA components of Reaction B of the one-tube BxPreaction described in Example 16, pEZC7102 and attB-tet-PCR.

FIG. 57 is a physical map of the desired product of Reaction B of theone-tube BxP reaction described in Example 16, tetx7102.

FIG. 58 is a physical map of the Destination Vector pEZC8402.

FIG. 59 is a physical map of the expected tet^(r) subclone product,tetx8402, resulting from the LxR Reaction with tetx7102 (FIG. 57) pluspEZC8402 (FIG. 58).

FIG. 60 is a schematic depiction of the bacteriophage lambdarecombination pathways in E. coli.

FIG. 61 is a schematic depiction of the DNA molecules participating inthe LR Reaction. Two different co-integrates form during the LR Reaction(only one of which is shown here), depending on whether attL1 and attR1or attL2 and attR2 are first to recombine. In one aspect, the inventionprovides directional cloning of a nucleic acid molecule of interest,since the recombination sites react with specificity (attL1 reacts withattR1; attL2 with attR2; attB1 with attP1; and attB2 with attP2). Thus,positioning of the sites allows construction of desired vectors havingrecombined fragments in the desired orientation.

FIG. 62 is a depiction of native and fusion protein expression using therecombinational cloning methods and compositions of the invention. Inthe upper figure depicting native protein expression, all of thetranslational start signals are included between the attB1 and attB2sites; therefore, these signals must be present in the starting EntryClone. The lower figure depicts fusion protein expression (here showingexpression with both N-terminal and C-terminal fusion tags so thatribosomes read through attB1 and attB2 to create the fusion protein).Unlike native protein expression vectors, N-terminal fusion vectors havetheir translational start signals upstream of the attB1 site.

FIG. 63 is a schematic depiction of three GATEWAY™ Cloning Systemcassettes. Three blunt-ended cassettes are depicted which convertstandard expression vectors to Destination Vectors. Each of the depictedcassettes provides amino-terminal fusions in one of three possiblereading frames, and each has a distinctive restriction cleavage site asshown.

FIG. 64 shows the physical maps of plasmids containing three attRreading frame cassettes, pEZC15101 (reading frame A; FIG. 64A),pEZC15102 (reading frame B; Figure MB), and pEZC15103 (reading frame C;FIG. 64C).

FIG. 65 depicts the attB primers used for amplifying the tee and amp^(r)genes from pBR322 bp the cloning methods of the invention.

FIG. 66 is a table listing the results of recombinational cloning of thetee and amp^(r) PCR products made using the primers shown in FIG. 65.

FIG. 67 is a graph showing the effect of the number of guanines (G's)contained on the 5′ end of the PCR primers on the cloning efficiency ofPCR products. It is noted, however, that other nucleotides besidesguanine (including A, T, C, U or combinations thereof) may be used as 5′extensions on the PCR primers to enhance cloning efficiency of PCRproducts.

FIG. 68 is a graph showing a titration of various amounts of attP andattB reactants in the BxP reaction, and the effects on cloningefficiency of PCR products.

FIG. 69 is a series of graphs showing the effects of various weights(FIG. 69A) or moles (FIG. 69B) of a 256 bp PCR product on formation ofcolonies, and on efficiency of cloning of the 256 bp PCR product into aDonor Vector (FIG. 69C).

FIG. 70 is a series of graphs showing the effects of various weights(FIG. 70A) or moles (FIG. 70B) of a 1 kb PCR product on formation ofcolonies, and on efficiency of cloning of the 1 kb PCR product into aDonor Vector (FIG. 70C).

FIG. 71 is a series of graphs showing the effects of various weights(FIG. 71A) or moles (FIG. 71B) of a 1.4 kb PCR product on formation ofcolonies, and on efficiency of cloning of the 1.4 kb PCR product into aDonor Vector (FIG. 71C).

FIG. 72 is a series of graphs showing the effects of various weights(FIG. 72A) or moles (FIG. 72B) of a 3.4 kb PCR product on formation ofcolonies, and on efficiency of cloning of the 3.4 kb PCR product into aDonor Vector (FIG. 72C).

FIG. 73 is a series of graphs showing the effects of various weights(FIG. 73A) or moles (FIG. 73B) of a 4.6 kb PCR product on formation ofcolonies, and on efficiency of cloning of the 4.6 kb PCR product into aDonor Vector (FIG. 73C).

FIG. 74 is photograph of an ethidium bromide-stained gel of a titrationof a 6.9 kb PCR product in a BxP reaction.

FIG. 75 is a graph showing the effects of various amounts of a 10.1 kbPCR product on formation of colonies upon cloning of the 10.1 kb PCRproduct into a Donor Vector.

FIG. 76 is photograph of an ethidium bromide-stained gel of a titrationof a 10.1 kb PCR product in a BxP reaction.

FIG. 77 is a table summarizing the results of the PCR product cloningefficiency experiments depicted in FIGS. 69-74, for PCR fragmentsranging in size from 0.256 kb to 6.9 kb.

FIG. 78 is a depiction of the sequences at the ends of attR Cassettes(SEQ ID NOs:163-170). Sequences contributed by the Cm^(r)-ccdB cassetteare shown, including the outer ends of the flanking attR sites (boxed).The staggered cleavage sites for Int are indicated in the boxed regions.Following recombination with an Entry Clone, only the outer sequences inattR sites contribute to the resulting attB sites in the ExpressionClone. The underlined sequences at both ends dictate the differentreading frames (reading frames A, B, or C, with two alternative readingframe C cassettes depicted) for fusion proteins.

FIG. 79 is a depiction of several different attR cassettes (SEQ IDNOs:171-173) (in reading frames A, B, or C) which may provide fusioncodons at the amino-terminus of the encoded protein.

FIG. 80 illustrates the single-cutting restriction sites in an attRreading frame A cassette of the invention.

FIG. 81 illustrates the single-cutting restriction sites in an attRreading frame B cassette of the invention.

FIG. 82 illustrates the single-cutting restriction sites in twoalternative attR reading frame C cassettes of the invention (FIGS. 82Aand 82B) depicted in FIG. 78.

FIG. 83 shows the physical map (FIG. 83A), and the nucleotide sequence(FIG. 83B-C) (SEQ ID NO:174), for an attR reading frame C parent plasmidprfC Parent III, which contains an attR reading frame C cassette of theinvention (alternative A in FIGS. 78 and 82).

FIG. 84 is a physical map of plasmid pEZC1301.

FIG. 85 is a physical map of plasmid pEZC1313.

FIG. 86 is a physical map of plasmid pEZ14032.

FIG. 87 is a physical map of plasmid pMAB58.

FIG. 88 is a physical map of plasmid pMAB62.

FIG. 89 is a depiction of a synthesis reaction using two pairs ofhomologous primers of the invention.

FIG. 90 is a schematic depiction of the physical map (FIG. 90A), and thenucleotide sequence (FIG. 90B-D) (SEQ ID NO:175), of Destination VectorpDEST28.

FIG. 91 is a schematic depiction of the physical map (FIG. 91A), and thenucleotide sequence (FIG. 91B-D) (SEQ ID NO:176), of Destination VectorpDEST29.

FIG. 92 is a schematic depiction of the physical map (FIG. 92A), and thenucleotide sequence (FIG. 92B-D) (SEQ ID NO:177), of Destination VectorpDEST30.

FIG. 93 is a schematic depiction of the physical map (FIG. 93A), and thenucleotide sequence (FIG. 93B-D) (SEQ ID NO:178), of Destination VectorpDEST31.

FIG. 94 is a schematic depiction of the physical map (FIG. 94A), and thenucleotide sequence (FIGS. 94B-F) (SEQ ID NO:179), of Destination VectorpDEST32.

FIG. 95 is a schematic depiction of the physical map (FIG. 95A), and thenucleotide sequence (FIGS. 95B-E) (SEQ ID NO:180), of Destination VectorpDEST33.

FIG. 96 is a schematic depiction of the physical map (FIG. 96A), and thenucleotide sequence (FIG. 96B-D) (SEQ ID NO:181), of Destination VectorpDEST34.

FIG. 97 is a depiction of the physical map (FIG. 97A), and thenucleotide sequence (FIG. 97B-C) (SEQ ID NO:182), for the Donor plasmidpDONR207 which donates a gentamycin-resistant vector in the BP Reaction.

FIG. 98 is a schematic depiction of the physical map (FIG. 98A), and thenucleotide sequence (FIG. 98B-D) (SEQ ID NO:183), of the 2-hybrid vectorpMAB85.

FIG. 99 is a schematic depiction of the physical map (FIG. 99A), and thenucleotide sequence (FIG. 99B-D) (SEQ ID NO:184), of the 2-hybrid vectorpMAB86.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the description that follows, a number of terms used in recombinantDNA technology are utilized extensively. In order to provide a clear andconsistent understanding of the specification and claims, including thescope to be given such terms, the following definitions are provided.

Byproduct: is a daughter molecule (a new clone produced after the secondrecombination event during the recombinational cloning process) lackingthe segment which is desired to be cloned or subcloned.

Cointegrate: is at least one recombination intermediate nucleic acidmolecule of the present invention that contains both parental (starting)molecules. It will usually be linear. In some embodiments it can becircular. RNA and polypeptides may be expressed from cointegrates usingan appropriate host cell strain, for example E. coli DB3.1 (particularlyE. coli LIBRARY EFFICIENCY® DB3.1™ Competent Cells), and selecting forboth selection markers found on the cointegrate molecule.

Host: is any prokaryotic or eukaryotic organism that can be a recipientof the recombinational cloning Product, vector, or nucleic acid moleculeof the invention. A “host,” as the term is used herein, includesprokaryotic or eukaryotic organisms that can be genetically engineered.For examples of such hosts, see Maniatis et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1982).

Insert or Inserts: include the desired nucleic acid segment or apopulation of nucleic acid segments (segment A of FIG. 1) which may bemanipulated by the methods of the present invention. Thus, the termsInsert(s) are meant to include a particular nucleic acid (preferablyDNA) segment or a population of segments. Such Insert(s) can compriseone or more nucleic acid molecules.

Insert Donor: is one of the two parental nucleic acid molecules (e.g.RNA or DNA) of the present invention which carries the Insert. TheInsert Donor molecule comprises the Insert flanked on both sides withrecombination sites. The Insert Donor can be linear or circular. In oneembodiment of the invention, the Insert Donor is a circular DNA moleculeand further comprises a cloning vector sequence outside of therecombination signals (see FIG. 1). When a population of Inserts orpopulation of nucleic acid segments are used to make the Insert Donor, apopulation of Insert Donors results and may be used in accordance withthe invention. Examples of such Insert Donor molecules are GATEWAY™Entry Vectors, which include but are not limited to those Entry Vectorsdepicted in FIGS. 10-20, as well as other vectors comprising a gene ofinterest flanked by one or more attL sites (e.g., attL1, attL2, etc.),or by one or more attB sites (e.g., attB1, attB2, etc.) for theproduction of library clones.

Product: is one of the desired daughter molecules comprising the A and Dsequences which is produced after the second recombination event duringthe recombinational cloning process (see FIG. 1). The Product containsthe nucleic acid which was to be cloned or subcloned. In accordance withthe invention, when a population of Insert Donors are used, theresulting population of Product molecules will contain all or a portionof the population of Inserts of the Insert Donors and preferably willcontain a representative population of the original molecules of theInsert Donors.

Promoter: is a DNA sequence generally described as the 5′-region of agene, located proximal to the start codon. The transcription of anadjacent DNA segment is initiated at the promoter region. A repressiblepromoter's rate of transcription decreases in response to a repressingagent. An inducible promoter's rate of transcription increases inresponse to an inducing agent. A constitutive promoter's rate oftranscription is not specifically regulated, though it can vary underthe influence of general metabolic conditions.

Recognition sequence: Recognition sequences are particular sequenceswhich a protein, chemical compound, DNA, or RNA molecule (e.g.,restriction endonuclease, a modification methylase, or a recombinase)recognizes and binds. In the present invention, a recognition sequencewill usually refer to a recombination site. For example, the recognitionsequence for Cre recombinase is loxP which is a 34 base pair sequencecomprised of two 13 base pair inverted repeats (serving as therecombinase binding sites) flanking an 8 base pair core sequence. SeeFIG. 1 of Sauer, B., Current Opinion in Biotechnology 5:521-527 (1994).Other examples of recognition sequences are the attB, attP, attL, andattR sequences which are recognized by the recombinase enzyme λIntegrase. attB is an approximately 25 base pair sequence containing two9 base pair core-type Int binding sites and a 7 base pair overlapregion. attP is an approximately 240 base pair sequence containingcore-type Int binding sites and arm-type hit binding sites as well assites for auxiliary proteins integration host factor (IHF), FIS andexcisionase (Xis). See Landy, Current Opinion in Biotechnology 3:699-707(1993). Such sites may also be engineered according to the presentinvention to enhance production of products in the methods of theinvention. When such engineered sites lack the P1 or H1 domains to makethe recombination reactions irreversible (e.g., attR or attP), suchsites may be designated attR′ or attP′ to show that the domains of thesesites have been modified in some way.

Recombination proteins: include excisive or integrative proteins,enzymes, co-factors or associated proteins that are involved inrecombination reactions involving one or more recombination sites, whichmay be wild-type proteins (See Landy, Current Opinion in Biotechnology3:699-707 (1993)), or mutants, derivatives (e.g., fusion proteinscontaining the recombination protein sequences or fragments thereof),fragments, and variants thereof.

Recombination site: is a recognition sequence on a DNA moleculeparticipating in an integration/recombination reaction by therecombinational cloning methods of the invention. Recombination sitesare discrete sections or segments of DNA on the participating nucleicacid molecules that are recognized and bound by a site-specificrecombination protein during the initial stages of integration orrecombination. For example, the recombination site for Cre recombinaseis loxP which is a 34 base pair sequence comprised of two 13 base pairinverted repeats (serving as the recombinase binding sites) flanking an8 base pair core sequence. See FIG. 1 of Sauer, B., Curr. Opin. Biotech.5:521-527 (1994). Other examples of recognition sequences include theattB, attP, attL, and attR sequences described herein, and mutants,fragments, variants and derivatives thereof, which are recognized by therecombination protein λ Int and by the auxiliary proteins integrationhost factor (IHF), FIS and excisionase (Xis). See Landy, Curr. Opin.Biotech. 3:699-707 (1993).

Recombinational Cloning: is a method described herein, whereby segmentsof nucleic acid molecules or populations of such molecules areexchanged, inserted, replaced, substituted or modified, in vitro or invivo. By “in vitro” and “in vivo” herein is meant recombinationalcloning that is carried out outside of host cells (e.g., in cell-freesystems) or inside of host cells (e.g., using recombination proteinsexpressed by host cells), respectively.

Repression cassette: is a nucleic acid segment that contains a repressoror a Selectable marker present in the subcloning vector.

Selectable marker: is a DNA segment that allows one to select for oragainst a molecule (e.g., a replicon) or a cell that contains it, oftenunder particular conditions. These markers can encode an activity, suchas, but not limited to, production of RNA, peptide, or protein, or canprovide a binding site for RNA, peptides, proteins, inorganic andorganic compounds or compositions and the like. Examples of Selectablemarkers include but are not limited to: (1) DNA segments that encodeproducts which provide resistance against otherwise toxic compounds(e.g., antibiotics); (2) DNA segments that encode products which areotherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophicmarkers); (3) DNA segments that encode products which suppress theactivity of a gene product; (4) DNA segments that encode products whichcan be readily identified (e.g., phenotypic markers such asβ-galactosidase, green fluorescent protein (GFP), and cell surfaceproteins); (5) DNA segments that bind products which are otherwisedetrimental to cell survival and/or function; (6) DNA segments thatotherwise inhibit the activity of any of the DNA segments described inNos. 1-5 above (e.g., antisense oligonucleotides); (7) DNA segments thatbind products that modify a substrate (e.g. restriction endonucleases);(8) DNA segments that can be used to isolate or identify a desiredmolecule (e.g. specific protein binding sites); (9) DNA segments thatencode a specific nucleotide sequence which can be otherwisenon-functional (e.g., for PCR amplification of subpopulations ofmolecules); (10) DNA segments, which when absent, directly or indirectlyconfer resistance or sensitivity to particular compounds; (11) DNAsegments that encode products which are toxic in recipient cells; (12)DNA segments that inhibit replication, partition or heritability ofnucleic acid molecules that contain them; and/or (13) DNA segments thatencode conditional replication functions, e.g., replication in certainhosts or host cell strains or under certain environmental conditions(e.g., temperature, nutritional conditions, etc.).

Selection scheme: is any method which allows selection, enrichment, oridentification of a desired Product or Product(s) from a mixturecontaining an Entry Clone or Vector, a Destination Vector, a DonorVector, an Expression Clone or Vector, any intermediates (e.g. aCointegrate or a replicon), and/or Byproducts. The selection schemes ofone preferred embodiment have at least two components that are eitherlinked or unlinked during recombinational cloning. One component is aSelectable marker. The other component controls the expression in vitroor in vivo of the Selectable marker, or survival of the cell (or thenucleic acid molecule, e.g., a replicon) harboring the plasmid carryingthe Selectable marker. Generally, this controlling element will be arepressor or inducer of the Selectable marker, but other means forcontrolling expression or activity of the Selectable marker can be used.Whether a repressor or activator is used will depend on whether themarker is for a positive or negative selection, and the exactarrangement of the various DNA segments, as will be readily apparent tothose skilled in the art. A preferred requirement is that the selectionscheme results in selection of or enrichment for only one or moredesired Products. As defined herein, selecting for a DNA moleculeincludes (a) selecting or enriching for the presence of the desired DNAmolecule, and (b) selecting or enriching against the presence of DNAmolecules that are not the desired DNA molecule.

In one embodiment, the selection schemes (which can be carried out inreverse) will take one of three forms, which will be discussed in termsof FIG. 1. The first, exemplified herein with a Selectable marker and arepressor therefore, selects for molecules having segment D and lackingsegment C. The second selects against molecules having segment C and formolecules having segment D. Possible embodiments of the second formwould have a DNA segment carrying a gene toxic to cells into which thein vitro reaction products are to be introduced. A toxic gene can be aDNA that is expressed as a toxic gene product (a toxic protein or RNA),or can be toxic in and of itself. (In the latter case, the toxic gene isunderstood to carry its classical definition of “heritable trait”.)

Examples of such toxic gene products are well known in the art, andinclude, but are not limited to, restriction endonucleases (e.g., DpnI),apoptosis-related genes (e.g. ASK1 or members of the bcl-2/ced-9family), retroviral genes including those of the human immunodeficiencyvirus (HIV), defensins such as NP-1, inverted repeats or pairedpalindromic DNA sequences, bacteriophage lytic genes such as those fromΦX174 or bacteriophage T4; antibiotic sensitivity genes such as rpsL,antimicrobial sensitivity genes such as pheS, plasmid killer genes,eukaryotic transcriptional vector genes that produce a gene producttoxic to bacteria, such as GATA-1, and genes that kill hosts in theabsence of a suppressing function, e.g., kicB, ccdB, ΦX174 E (Liu, Q. etal., Curr. Biol. 8:1300-1309 (1998)), and other genes that negativelyaffect replicon stability and/or replication. A toxic gene canalternatively be selectable in vitro, e.g., a restriction site.

Many genes coding for restriction endonucleases operably linked toinducible promoters are known, and may be used in the present invention.See, e.g. U.S. Pat. No. 4,960,707 (DpnI and DpnII); U.S. Pat. Nos.5,000,333, 5,082,784 and 5,192,675 (KpnI); U.S. Pat. No. 5,147,800(NgoAIII and NgoAI); U.S. Pat. No. 5,179,015 (FspI and HaeIII): U.S.Pat. No. 5,200,333 (HaeII and TaqI); U.S. Pat. No. 5,248,605 (HpaII);U.S. Pat. No. 5,312,746 (ClaI); U.S. Pat. No. 5,231,021 and U.S. Pat.No. 5,304,480 (XhoI and XhoII); U.S. Pat. No. 5,334,526 (AluI); U.S.Pat. No. 5,470,740 (NsiI); U.S. Pat. No. 5,534,428 (SstI/SacI); U.S.Pat. No. 5,202,248 (NcoI); U.S. Pat. No. 5,139,942 (NdeI); and U.S. Pat.No. 5,098,839 (PacI). See also Wilson, G. G., Nucl. Acids Res.19:2539-2566 (1991); and Lunnen, K. D., et al., Gene 74:25-32 (1988).

In the second form, segment D carries a Selectable marker. The toxicgene would eliminate transformants harboring the Vector Donor,Cointegrate, and Byproduct molecules, while the Selectable marker can beused to select for cells containing the Product and against cellsharboring only the Insert Donor.

The third form selects for cells that have both segments A and D in cison the same molecule, but not for cells that have both segments in transon different molecules. This could be embodied by a Selectable markerthat is split into two inactive fragments, one each on segments A and D.

The fragments are so arranged relative to the recombination sites thatwhen the segments are brought together by the recombination event, theyreconstitute a functional Selectable marker. For example, therecombinational event can link a promoter with a structural nucleic acidmolecule (e.g., a gene), can link two fragments of a structural nucleicacid molecule, or can link nucleic acid molecules that encode aheterodimeric gene product needed for survival, or can link portions ofa replicon.

Site-specific recombinase: is a type of recombinase which typically hasat least the following four activities (or combinations thereof): (1)recognition of one or two specific nucleic acid sequences; (2) cleavageof said sequence or sequences; (3) topoisomerase activity involved instrand exchange; and (4) ligase activity to reseal the cleaved strandsof nucleic acid. See Sauer, B., Current Opinions in Biotechnology5:521-527 (1994). Conservative site-specific recombination isdistinguished from homologous recombination and transposition by a highdegree of sequence specificity for both partners. The strand exchangemechanism involves the cleavage and rejoining of specific DNA sequencesin the absence of DNA synthesis (Landy, A. (1989) Ann. Rev. Biochem.58:913-949).

Subcloning vector: is a cloning vector comprising a circular or linearnucleic acid molecule which includes preferably an appropriate replicon.In the present invention, the subcloning vector (segment D in FIG. 1)can also contain functional and/or regulatory elements that are desiredto be incorporated into the final product to act upon or with the clonedDNA Insert (segment A in FIG. 1). The subcloning vector can also containa Selectable marker (preferably DNA).

Vector: is a nucleic acid molecule (preferably DNA) that provides auseful biological or biochemical property to an Insert. Examples includeplasmids, phages, autonomously replicating sequences (ARS), centromeres,and other sequences which are able to replicate or be replicated invitro or in a host cell, or to convey a desired nucleic acid segment toa desired location within a host cell. A Vector can have one or morerestriction endonuclease recognition sites at which the sequences can becut in a determinable fashion without loss of an essential biologicalfunction of the vector, and into which a nucleic acid fragment can bespliced in order to bring about its replication and cloning. Vectors canfurther provide primer sites, e.g., for PCR, transcriptional and/ortranslational initiation and/or regulation sites, recombinationalsignals, replicons, Selectable markers, etc. Clearly, methods ofinserting a desired nucleic acid fragment which do not require the useof homologous recombination, transpositions or restriction enzymes (suchas, but not limited to, UDG cloning of PCR fragments (U.S. Pat. No.5,334,575, entirely incorporated herein by reference), T:A cloning, andthe like) can also be applied to clone a fragment into a cloning vectorto be used according to the present invention. The cloning vector canfurther contain one or more selectable markers suitable for use in theidentification of cells transformed with the cloning vector.

Vector Donor: is one of the two parental nucleic acid molecules (e.g.RNA or DNA) of the present invention which carries the DNA segmentscomprising the DNA vector which is to become part of the desiredProduct. The Vector Donor comprises a subcloning vector D (or it can becalled the cloning vector if the Insert Donor does not already contain acloning vector (e.g., for PCR fragments containing attB sites; seebelow)) and a segment C flanked by recombination sites (see FIG. 1).Segments C and/or D can contain elements that contribute to selectionfor the desired Product daughter molecule, as described above forselection schemes. The recombination signals can be the same ordifferent, and can be acted upon by the same or different recombinases.In addition, the Vector Donor can be linear or circular. Examples ofsuch Vector Donor molecules include GATEWAY™ Destination Vectors, whichinclude but are not limited to those Destination Vectors depicted inFIGS. 21-47 and 90-96.

Primer: refers to a single stranded or double stranded oligonucleotidethat is extended by covalent bonding of nucleotide monomers duringamplification or polymerization of a nucleic acid molecule (e.g. a DNAmolecule). In a preferred aspect, a primer comprises one or morerecombination sites or portions of such recombination sites. Portions ofrecombination sites comprise at least 2 bases (or basepairs, abbreviatedherein as “bp”), at least 5-200 bases, at least 10-100 bases, at least15-75 bases, at least 15-50 bases, at least 15-25 bases, or at least16-25 bases, of the recombination sites of interest, as described infurther detail below and in the Examples. When using portions ofrecombination sites, the missing portion of the recombination site maybe provided as a template by the newly synthesized nucleic acidmolecule. Such recombination sites may be located within and/or at oneor both termini of the primer. Preferably, additional sequences areadded to the primer adjacent to the recombination site(s) to enhance orimprove recombination and/or to stabilize the recombination site duringrecombination. Such stabilization sequences may be any sequences(preferably G/C rich sequences) of any length. Preferably, suchsequences range in size from 1 to about 1000 bases, 1 to about 500bases, and 1 to about 100 bases, 1 to about 60 bases, 1 to about 25, 1to about 10, 2 to about 10 and preferably about 4 bases. Preferably,such sequences are greater than 1 base in length and preferably greaterthan 2 bases in length.

Template: refers to double stranded or single stranded nucleic acidmolecules which are to be amplified, synthesized or sequenced. In thecase of double stranded molecules, denaturation of its strands to form afirst and a second strand is preferably performed before these moleculeswill be amplified, synthesized or sequenced, or the double strandedmolecule may be used directly as a template. For single strandedtemplates, a primer complementary to a portion of the template ishybridized under appropriate conditions and one or more polypeptideshaving polymerase activity (e.g. DNA polymerases and/or reversetranscriptases) may then synthesize a nucleic acid moleculecomplementary to all or a portion of said template. Alternatively, fordouble stranded templates, one or more promoters may be used incombination with one or more polymerases to make nucleic acid moleculescomplementary to all or a portion of the template. The newly synthesizedmolecules, according to the invention, may be equal or shorter in lengththan the original template. Additionally, a population of nucleic acidtemplates may be used during synthesis or amplification to produce apopulation of nucleic acid molecules typically representative of theoriginal template population.

Adapter: is an oligonucleotide or nucleic acid fragment or segment(preferably DNA) which comprises one or more recombination sites (orportions of such recombination sites) which in accordance with theinvention can be added to a circular or linear Insert Donor molecule aswell as other nucleic acid molecules described herein. When usingportions of recombination sites, the missing portion may be provided bythe Insert Donor molecule. Such adapters may be added at any locationwithin a circular or linear molecule, although the adapters arepreferably added at or near one or both termini of a linear molecule.Preferably, adapters are positioned to be located on both sides(flanking) a particular nucleic acid molecule of interest. In accordancewith the invention, adapters may be added to nucleic acid molecules ofinterest by standard recombinant techniques (e.g. restriction digest andligation). For example, adapters may be added to a circular molecule byfirst digesting the molecule with an appropriate restriction enzyme,adding the adapter at the cleavage site and reforming the circularmolecule which contains the adapter(s) at the site of cleavage. In otheraspects, adapters may be added by homologous recombination, byintegration of RNA molecules, and the like. Alternatively, adapters maybe ligated directly to one or more and preferably both termini of alinear molecule thereby resulting in linear molecule(s) having adaptersat one or both termini. In one aspect of the invention, adapters may beadded to a population of linear molecules, (e.g. a cDNA library orgenomic DNA which has been cleaved or digested) to form a population oflinear molecules containing adapters at one and preferably both terminiof all or substantial portion of said population.

Adapter-Primer: is primer molecule which comprises one or morerecombination sites (or portions of such recombination sites) which inaccordance with the invention can be added to a circular or linearnucleic acid molecule described herein. When using portions ofrecombination sites, the missing portion may be provided by a nucleicacid molecule (e.g., an adapter) of the invention. Such adapter-primersmay be added at any location within a circular or linear molecule,although the adapter-primers are preferably added at or near one or bothtermini of a linear molecule. Examples of such adapter-primers and theuse thereof in accordance with the methods of the invention are shown inExample 25 herein. Such adapter-primers may be used to add one or morerecombination sites or portions thereof to circular or linear nucleicacid molecules in a variety of contexts and by a variety of techniques,including but not limited to amplification (e.g., PCR), ligation (e.g.,enzymatic or chemical/synthetic ligation), recombination (e.g.,homologous or non-homologous (illegitimate) recombination) and the like.

Library: refers to a collection of nucleic acid molecules (circular orlinear). In one embodiment, a library may comprise a plurality (i.e.,two or more) of DNA molecules, which may or may not be from a commonsource organism, organ, tissue, or cell. In another embodiment, alibrary is representative of all or a portion or a significant portionof the DNA content of an organism (a “genomic” library), or a set ofnucleic acid molecules representative of all or a portion or asignificant portion of the expressed nucleic acid molecules (a cDNAlibrary) in a cell, tissue, organ or organism. A library may alsocomprise random sequences made by de novo synthesis, mutagenesis of oneor more sequences and the like. Such libraries may or may not becontained in one or more vectors.

Amplification: refers to any in vitro method for increasing a number ofcopies of a nucleotide sequence with the use of a polymerase. Nucleicacid amplification results in the incorporation of nucleotides into aDNA and/or RNA molecule or primer thereby forming a new moleculecomplementary to a template. The formed nucleic acid molecule and itstemplate can be used as templates to synthesize additional nucleic acidmolecules. As used herein, one amplification reaction may consist ofmany rounds of replication. DNA amplification reactions include, forexample, polymerase chain reaction (PCR). One PCR reaction may consistof 5-100 “cycles” of denaturation and synthesis of a DNA molecule.

Oligonucleotide: refers to a synthetic or natural molecule comprising acovalently linked sequence of nucleotides which are joined by aphosphodiester bond between the 3′ position of the deoxyribose or riboseof one nucleotide and the 5′ position of the deoxyribose or ribose ofthe adjacent nucleotide. This term may be used interchangeably hereinwith the terms “nucleic acid molecule” and “polynucleotide,” without anyof these terms necessarily indicating any particular length of thenucleic acid molecule to which the term specifically refers.

Nucleotide: refers to a base-sugar-phosphate combination. Nucleotidesare monomeric units of a nucleic acid molecule (DNA and RNA). The termnucleotide includes ribonucleoside triphosphates ATP, UTP, CTG, GTP anddeoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP,dTTP, or derivatives thereof. Such derivatives include, for example,[αS]dATP, 7-deaza-dGTP and 7-deaza-dATP. The term nucleotide as usedherein also refers to dideoxyribonucleoside triphosphates (ddNTPs) andtheir derivatives. Illustrated examples of dideoxyribonucleosidetriphosphates include, but are not limited to, ddATP, ddCTP, ddGTP,ddITP, and ddTTP. According to the present invention, a “nucleotide” maybe unlabeled or detectably labeled by well known techniques. Detectablelabels include, for example, radioactive isotopes, fluorescent labels,chemiluminescent labels, bioluminescent labels and enzyme labels.

Hybridization: The terms “hybridization” and “hybridizing” refers tobase pairing of two complementary single-stranded nucleic acid molecules(RNA and/or DNA) to give a double stranded molecule. As used herein, twonucleic acid molecules may be hybridized, although the base pairing isnot completely complementary. Accordingly, mismatched bases do notprevent hybridization of two nucleic acid molecules provided thatappropriate conditions, well known in the art, are used. In someaspects, hybridization is said to be under “stringent conditions.” By“stringent conditions” as used herein is meant overnight incubation at42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 g/ml denatured, sheared salmonsperm DNA, followed by washing the filters in 0.1×SSC at about 65° C.

Other terms used in the fields of recombinant DNA technology andmolecular and cell biology as used herein will be generally understoodby one of ordinary skill in the applicable arts.

Overview

Two reactions constitute the recombinational cloning system of thepresent invention, referred to herein as the “GATEWAY™ Cloning System,”as depicted generally in FIG. 1. The first of these reactions, the LRReaction (FIG. 2), which may also be referred to interchangeably hereinas the Destination Reaction, is the main pathway of this system. The LRReaction is a recombination reaction between an Entry vector or cloneand a Destination Vector, mediated by a cocktail of recombinationproteins such as the GATEWAY™ LR Clonase™ Enzyme Mix described herein.This reaction transfers nucleic acid molecules of interest (which may begenes, cDNAs, cDNA libraries, or fragments thereof) from the Entry Cloneto an Expression Vector, to create an Expression Clone.

The sites labeled L, R, B, and P are respectively the attL, attR, attB,and attP recombination sites for the bacteriophage λ recombinationproteins that constitute the Clonase cocktail (referred to hereinvariously as “Clonase” or “GATEWAY™ LR Clonase™ Enzyme Mix” (forrecombination protein mixtures mediating attL×attR recombinationreactions, as described herein) or “GATEWAY™ BP Clonase™ Enzyme Mix”(for recombination protein mixtures mediating attB×attP recombinationreactions, as described herein)). The Recombinational Cloning reactionsare equivalent to concerted, highly specific, cutting and ligationreactions. Viewed in this way, the recombination proteins cut to theleft and right of the nucleic acid molecule of interest in the EntryClone and ligate it into the Destination vector, creating a newExpression Clone.

The nucleic acid molecule of interest in an Expression Clone is flankedby the small attB1 and attB2 sites. The orientation and reading frame ofthe nucleic acid molecule of interest are maintained throughout thesubcloning, because attL1 reacts only with attR1, and attL2 reacts onlywith attR2. Likewise, attB1 reacts only with attP1, and attB2 reactsonly with attP2. Thus, the invention also relates to methods ofcontrolled or directional cloning using the recombination sites of theinvention (or portions thereof), including variants, fragments, mutantsand derivatives thereof which may have altered or enhanced specificity.The invention also relates more generally to any number of recombinationsite partners or pairs (where each recombination site is specific forand interacts with its corresponding recombination site). Suchrecombination sites are preferably made by mutating or modifying therecombination site to provide any number of necessary specificities(e.g., attB1-10, attP1-10, attL1-10, attR1-10, etc.), non-limitingexamples of which are described in detail in the Examples herein.

When an aliquot from the recombination reaction is transformed into hostcells (e.g., E. coli) and spread on plates containing an appropriateselection agent, e.g., an antibiotic such as ampicillin with or withoutmethicillin, cells that take up the desired clone form colonies. Theunreacted Destination Vector does not give ampicillin-resistantcolonies, even though it carries the ampicillin-resistance gene, becauseit contains a toxic gene, e.g., ccdB. Thus selection for ampicillinresistance selects for E. coli cells that carry the desired product,which usually comprise >90% of the colonies on the ampicillin plate.

To participate in the Recombinational (or “GATEWAY™”) Cloning Reaction,a nucleic acid molecule of interest first may be cloned into an EntryVector, creating an Entry Clone. Multiple options are available forcreating Entry Clones, including: cloning of PCR sequences with terminalattB recombination sites into Entry Vectors; using the GATEWAY™ CloningSystem recombination reaction; transfer of genes from libraries preparedin GATEWAY™ Cloning System vectors by recombination into Entry Vectors;and cloning of restriction enzyme-generated fragments and PCR fragmentsinto Entry Vectors by standard recombinant DNA methods. These approachesare discussed in further detail herein.

A key advantage of the GATEWAY™ Cloning System is that a nucleic acidmolecule of interest (or even a population of nucleic acid molecules ofinterest) present as an Entry Clone can be subcloned in parallel intoone or more Destination Vectors in a simple reactions for anywhere fromabout 30 seconds to about 60 minutes (preferably about 1-60 minutes,about 1-45 minutes, about 1-30 minutes, about 2-60 minutes, about 2-45minutes, about 2-30 minutes, about 1-2 minutes, about 30-60 minutes,about 45-60 minutes, or about 30-45 minutes). Longer reaction times(e.g., 2-24 hours, or overnight) may increase recombination efficiency,particularly where larger nucleic acid molecules are used, as describedin the Examples herein. Moreover, a high percentage of the coloniesobtained carry the desired Expression Clone. This process is illustratedschematically in FIG. 3, which shows an advantage of the invention inwhich the molecule of interest can be moved simultaneously or separatelyinto multiple Destination Vectors. In the LR Reaction, one or both ofthe nucleic acid molecules to be recombined may have any topology (e.g.,linear, relaxed circular, nicked circular, supercoiled, etc.), althoughone or both are preferably linear.

The second major pathway of the GATEWAY™ Cloning System is the BPReaction (FIG. 4), which may also be referred to interchangeably hereinas the Entry Reaction or the Gateward Reaction. The BP Reaction mayrecombine an Expression Clone with a Donor Plasmid (the counterpart ofthe byproduct in FIG. 2). This reaction transfers the nucleic acidmolecule of interest (which may have any of a variety of topologies,including linear, coiled, supercoiled, etc.) in the Expression Cloneinto an Entry Vector, to produce a new Entry Clone. Once this nucleicacid molecule of interest is cloned into an Entry Vector, it can betransferred into new Expression Vectors, through the LR Reaction asdescribed above. In the BP Reaction, one or both of the nucleic acidmolecules to be recombined may have any topology (e.g., linear, relaxedcircular, nicked circular, supercoiled, etc.), although one or both arepreferably linear.

A useful variation of the BP Reaction permits rapid cloning andexpression of products of amplification (e.g., PCR) or nucleic acidsynthesis. Amplification (e.g., PCR) products synthesized with primerscontaining terminal 25 bp attB sites serve as efficient substrates forthe Gateward Cloning reaction. Such amplification products may berecombined with a Donor Vector to produce an Entry Clone (see FIG. 7).The result is an Entry Clone containing the amplification fragment. SuchEntry Clones can then be recombined with Destination Vectors—through theLR Reaction—to yield Expression Clones of the PCR product.

Additional details of the LR Reaction are shown in FIG. 5A. The GATEWAY™LR Clonase™ Enzyme Mix that mediates this reaction contains lambdarecombination proteins Int (Integrase), Xis (Excisionase), and IHF(Integration Host Factor). In contrast, the GATEWAY™ BP Clonase™ EnzymeMix, which mediates the BP Reaction (FIG. 5B), comprises Int and IHFalone.

The recombination (att) sites of each vector comprise two distinctsegments, donated by the parental vectors. The staggered lines dividingthe two portions of each att site, depicted in FIGS. 5A and 5B,represent the seven-base staggered cut produced by Int during therecombination reactions. This structure is seen in greater detail inFIG. 6, which displays the attB recombination sequences of an ExpressionClone, generated by recombination between the attL1 and attL2 sites ofan Entry Clone and the attR1 and attR2 sites of a Destination Vector.

The nucleic acid molecule of interest in the Expression Clone is flankedby attB sites: attB1 to the left (amino terminus) and attB2 to the right(carboxy terminus). The bases in attB1 to the left of the seven-basestaggered cut produced by Int are derived from the Destination vector,and the bases to the right of the staggered cut are derived from theEntry Vector (see FIG. 6). Note that the sequence is displayed intriplets corresponding to an open reading frame. If the reading frame ofthe nucleic acid molecule of interest cloned in the Entry Vector is inphase with the reading frame shown for attB1, amino-terminal proteinfusions can be made between the nucleic acid molecule of interest andany GATEWAY™ Cloning System Destination Vector encoding anamino-terminal fusion domain. Entry Vectors and Destination Vectors thatenable cloning in all three reading frames are described in more detailherein, particularly in the Examples.

The LR Reaction allows the transfer of a desired nucleic acid moleculeof interest into new Expression Vectors by recombining a Entry Clonewith various Destination Vectors. To participate in the LR orDestination Reaction, however, a nucleic acid molecule of interestpreferably is first converted to a Entry Clone. Entry Clones can be madein a number of ways, as shown in FIG. 7.

One approach is to clone the nucleic acid molecule of interest into oneor more of the Entry Vectors, using standard recombinant DNA methods,with restriction enzymes and ligase. The starting DNA fragment can begenerated by restriction enzyme digestion or as a PCR product. Thefragment is cloned between the attL1 and attL2 recombination sites inthe Entry Vector. Note that a toxic or “death” gene (e.g., ccdB),provided to minimize background colonies from incompletely digestedEntry Vector, must be excised and replaced by the nucleic acid moleculeof interest.

A second approach to making an Entry Clone (FIG. 7) is to make a library(genomic or cDNA) in an Entry Vector, as described in detail herein.Such libraries may then be transferred into Destination Vectors forexpression screening, for example in appropriate host cells such asyeast cells or mammalian cells.

A third approach to making Entry Clones (FIG. 7) is to use ExpressionClones obtained from cDNA molecules or libraries prepared in ExpressionVectors. Such cDNAs or libraries, flanked by attB sites, can beintroduced into a Entry. Vector by recombination with a Donor Vector viathe BP Reaction. If desired, an entire Expression Clone library can betransferred into the Entry Vector through the BP Reaction. ExpressionClone cDNA libraries may also be constructed in a variety of prokaryoticand eukaryotic GATEWAY™-modified vectors (e.g., the pEXP501 ExpressionVector (see FIG. 48), and 2-hybrid and attB library vectors), asdescribed in detail herein, particularly in the Examples below.

A fourth, and potentially most versatile, approach to making an EntryClone (FIG. 7) is to introduce a sequence for a nucleic acid molecule ofinterest into an Entry Vector by amplification (e.g., PCR) fragmentcloning. This method is diagramed in FIG. 8. The DNA sequence first isamplified (for example, with PCR) as outlined in detail below and in theExamples herein, using primers containing one or more bp, two or morebp, three or more bp, four or more bp, five or more bp, preferably sixor more bp, more preferably 6-25 bp (particularly 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24 or 25) by of the attB nucleotidesequences (such as, but not limited to, those depicted in FIG. 9), andoptionally one or more, two or more, three or more, four or more, andmost preferably four or five or more additional terminal nucleotidebases which preferably are guanines. The PCR product then may beconverted to a Entry Clone by performing a BP Reaction, in which theattB-PCR product recombines with a Donor Vector containing one or moreattP sites. Details of this approach and protocols for PCR fragmentsubcloning are provided in Examples 8 and 21-25.

A variety of Entry Clones may be produced by these methods, providing awide array of cloning options; a number of specific Entry Vectors arealso available commercially from Life Technologies, Inc. (Rockville,Md.). The Examples herein provide a more in-depth description ofselected Entry Vectors and details of their cloning sites. Choosing theoptimal Entry Vector for a particular application is discussed inExample 4.

Entry Vectors and Destination Vectors should be constructed so that theamino-terminal region of a nucleic acid molecule of interest (e.g., agene, cDNA library or insert, or fragment thereof) will be positionednext to the attL1 site. Entry Vectors preferably contain the rrnBtranscriptional terminator upstream of the attL1 site. This sequenceensures that expression of cloned nucleic acid molecules of interest isreliably “off” in E. coli, so that even toxic genes can be successfullycloned. Thus, Entry Clones may be designed to be transcriptionallysilent. Note also that Entry Vectors, and hence Entry Clones, maycontain the kanamycin antibiotic resistance (kan^(r)) gene to facilitateselection of host cells containing Entry Clones after transformation. Incertain applications, however, Entry Clones may contain other selectionmarkers, including but not limited to a gentamycin resistance (gen^(r))or tetracycline resistance (tet^(r)) gene, to facilitate selection ofhost cells containing Entry Clones after transformation.

Once a nucleic acid molecule of interest has been cloned into an EntryVector, it may be moved into a Destination Vector. The upper rightportion of FIG. 5A shows a schematic of a Destination Vector. The thickarrow represents some function (often transcription or translation) thatwill act on the nucleic acid molecule of interest in the clone. Duringthe recombination reaction, the region between the attR1 and attR2sites, including a toxic or “death” gene (e.g., ccdB), is replaced bythe DNA segment from the Entry Clone. Selection for recombinants thathave acquired the ampicillin resistance (amp^(r)) gene (carried on theDestination Vector) and that have also lost the death gene ensures thata high percentage (usually >90%) of the resulting colonies will containthe correct insert.

To move a nucleic acid molecule of interest into a Destination Vector,the Destination Vector is mixed with the Entry Clone comprising thedesired nucleic acid molecule of interest, a cocktail of recombinationproteins (e.g., GATEWAY™ LR Clonase™ Enzyme Mix) is added, the mixtureis incubated (preferably at about 25° C. for about 60 minutes, or longerunder certain circumstances, e.g. for transfer of large nucleic acidmolecules, as described below) and any standard host cell (includingbacterial cells such as E. coli; animal cells such as insect cells,mammalian cells, nematode cells and the like; plant cells; and yeastcells) strain is transformed with the reaction mixture. The host cellused will be determined by the desired selection (e.g., E. coli DB3.1,available commercially from Life Technologies, Inc., allows survival ofclones containing the ccdB death gene, and thus can be used to selectfor cointegrate molecules—i.e., molecules that are hybrids between theEntry Clone and Destination Vector). The Examples below provide furtherdetails and protocols for use of Entry and Destination Vectors intransferring nucleic acid molecules of interest and expressing RNAs orpolypeptides encoded by these nucleic acid molecules in a variety ofhost cells.

The cloning system of the invention therefore offers multipleadvantages:

-   -   Once a nucleic acid molecule of interest is cloned into the        GATEWAY™ Cloning System, it can be moved into and out of other        vectors with complete fidelity of reading frame and orientation.        That is, since the reactions proceed whereby attL1 on the Entry        Clone recombines with attR1 on the Destination Vector, the        directionality of the nucleic acid molecule of interest is        maintained or may be controlled upon transfer from the Entry        Clone into the Destination Vector. Hence, the GATEWAY™ Cloning        System provides a powerful and easy method of directional        cloning of nucleic acid molecule of interest.    -   One-step cloning or subcloning: Mix the Entry Clone and the        Destination Vector with Clonase, incubate, and transform.    -   Clone PCR products readily by in vitro recombination, by adding        attB sites to PCR primers. Then directly transfer these Entry        Clones into Destination Vectors. This process may also be        carried out in one step (see Examples below).    -   Powerful selections give high reliability: >90% (and often >99%)        of the colonies contain the desired DNA in its new vector.    -   One-step conversion of existing standard vectors into GATEWAY™        Cloning System vectors.    -   Ideal for large vectors or those with few cloning sites.    -   Recombination sites are short (25 bp), and may be engineered to        contain no stop codons or secondary structures.    -   Reactions may be automated, for high-throughput applications        (e.g., for diagnostic purposes or for therapeutic candidate        screening).    -   The reactions are economical: 0.3 μg of each DNA; no restriction        enzymes, phosphatase, ligase, or gel purification. Reactions        work well with miniprep DNA.    -   Transfer multiple clones, and even libraries, into one or more        Destination Vectors, in a single experiment.    -   A variety of Destination Vectors may be produced, for        applications including, but not limited to:        -   Protein expression in E. coli: native proteins; fusion            proteins with GST, His6, thioredoxin, etc., for            purification, or one or more epitope tags; any promoter            useful in expressing proteins in E. coli may be used, such            as ptrc, λP_(L), and T7 promoters.        -   Protein expression in eukaryotic cells: CMV promoter,            baculovirus (with or without His6 tag), Semliki Forest            virus, Tet regulation.        -   DNA sequencing (all lac primers), RNA probes, phagemids            (both strands)    -   A variety of Entry Vectors (for recombinational cloning entry by        standard recombinant DNA methods) may be produced:        -   Strong transcription stop just upstream, for genes toxic            to E. coli.        -   Three reading frames.        -   With or without TEV protease cleavage site.        -   Motifs for prokaryotic and/or eukaryotic translation.        -   Compatible with commercial cDNA libraries.    -   Expression Clone cDNA (attB) libraries, for expression        screening, including 2-hybrid libraries and phage display        libraries, may also be constructed.

Recombination Site Sequences

In one aspect, the invention relates to nucleic acid molecules, whichmay or may not be isolated nucleic acid molecules, comprising one ormore nucleotide sequences encoding one or more recombination sites orportions thereof. In particular, this aspect of the invention relates tosuch nucleic acid molecules comprising one or more nucleotide sequencesencoding attB, attP, attL, or attR, or portions of these recombinationsite sequences. The invention also relates to mutants, derivatives, andfragments of such nucleic acid molecules. Unless otherwise indicated,all nucleotide sequences that may have been determined by sequencing aDNA molecule herein were determined using manual or automated DNAsequencing, such as dideoxy sequencing, according to methods that areroutine to one of ordinary skill in the art (Sanger, F., and Coulson, A.R., J. Mol. Biol. 94:444-448 (1975); Sanger, F., et al., Proc. Natl.Acad. Sci. USA 74:5463-5467 (1977)). All amino acid sequences ofpolypeptides encoded by DNA molecules determined herein were predictedby conceptual translation of a DNA sequence determined as above.Therefore, as is known in the art for any DNA sequence determined bythese approaches, any nucleotide sequence determined herein may containsome errors. Nucleotide sequences determined by such methods aretypically at least about 90% identical, more typically at least about95% to at least about 99.9% identical to the actual nucleotide sequenceof the sequenced DNA molecule. As is also known in the art, a singleinsertion or deletion in a determined nucleotide sequence compared tothe actual sequence will cause a frame shift in translation of thenucleotide sequence such that the predicted amino acid sequence encodedby a determined nucleotide sequence will be completely different fromthe amino acid sequence actually encoded by the sequenced DNA molecule,beginning at the point of such an insertion or deletion.

Unless otherwise indicated, each “nucleotide sequence” set forth hereinis presented as a sequence of deoxyribonucleotides (abbreviated A, G, Cand T). However, by “nucleotide sequence” of a nucleic acid molecule orpolynucleotide is intended, for a DNA molecule or polynucleotide, asequence of deoxyribonucleotides, and for an RNA molecule orpolynucleotide, the corresponding sequence of ribonucleotides (A, G, Cand U), where each thymidine deoxyribonucleotide (T) in the specifieddeoxyribonucleotide sequence is replaced by the ribonucleotide uridine(U). Thus, the invention relates to sequences of the invention in theform of DNA or RNA molecules, or hybrid DNA/RNA molecules, and theircorresponding complementary DNA, RNA, or DNA/RNA strands.

In a first such aspect, the invention provides nucleic acid moleculescomprising one or more nucleotide sequences encoding attB1, or mutants,fragments, variants or derivatives thereof. Such nucleic acid moleculesmay comprise an attB1 nucleotide sequence having the sequence set forthin FIG. 9, such as: ACAAGTTTGTACAAAAAAGCAGGCT (SEQ ID NO:1), or anucleotide sequence complementary to the nucleotide sequence set forthin FIG. 9 for attB1, or mutants, fragments, variants or derivativesthereof. As one of ordinary skill will appreciate, however, certainmutations, insertions, or deletions of one or more bases in the attB1sequence contained in the nucleic acid molecules of the invention may bemade without compromising the structural and functional integrity ofthese molecules; hence, nucleic acid molecules comprising suchmutations, insertions, or deletions in the attB1 sequence areencompassed within the scope of the invention.

In a related aspect, the invention provides nucleic acid moleculescomprising one or more nucleotide sequences encoding attB2, or mutants,fragments, variants or derivatives thereof. Such nucleic acid moleculesmay comprise an attB2 nucleotide sequence having the sequence set forthin FIG. 9, such as: ACCCAGCTTTCTTGTACAAAGTGGT (SEQ ID NO:2), or anucleotide sequence complementary to the nucleotide sequence set forthin FIG. 9 for attB2, or mutants, fragments, variants or derivativesthereof. As noted above for attB1, certain mutations, insertions, ordeletions of one or more bases in the attB2 sequence contained in thenucleic acid molecules of the invention may be made without compromisingthe structural and functional integrity of these molecules; hence,nucleic acid molecules comprising such mutations, insertions, ordeletions in the attB2 sequence are encompassed within the scope of theinvention.

A recombinant host cell comprising a nucleic acid molecule containingattB1 and attB2 sites (the vector pEXP501, also known as pCMVSport6; seeFIG. 48), E. coli DB3.1(pCMVSport6), was deposited on Feb. 27, 1999,with the Collection, Agricultural Research Culture Collection (NRRL),1815 North University Street, Peoria, Ill. 61604 USA, as Deposit No.NRRL B-30108. The attB1 and attB2 sites within the deposited nucleicacid molecule are contained in nucleic acid cassettes in associationwith one or more additional functional sequences as described in moredetail below.

In another related aspect, the invention provides nucleic acid moleculescomprising one or more nucleotide sequences encoding attP1, or mutants,fragments, variants or derivatives thereof. Such nucleic acid moleculesmay comprise an attP1 nucleotide sequence having the sequence set forthin FIG. 9, such as:TACAGGTCACTAATACCATCTAAGTAGTTGATTCATAGTGA-CTGGATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTAT-GCAAAATCTAATTTAATATATTGATATTTATATCATTTTACGTT-TCTCGTTCAGCTTTTTTGTACAAAGTTGGCATTATAAAAAAGCATTG-CTCATCAATTTGTTGCAACGAACAGGTCACTATCAGTCAAAATAA-AATCATTATTTG(SEQ ID NO:3), or a nucleotide sequence complementary to the nucleotidesequence set forth in FIG. 9 for attP1, or mutants, fragments, variantsor derivatives thereof. As noted above for attB1, certain mutations,insertions, or deletions of one or more bases in the attP1 sequencecontained in the nucleic acid molecules of the invention may be madewithout compromising the structural and functional integrity of thesemolecules; hence, nucleic acid molecules comprising such mutations,insertions, or deletions in the attP1 sequence are encompassed withinthe scope of the invention.

In another related aspect, the invention provides nucleic acid moleculescomprising one or more nucleotide sequences encoding attP2, or mutants,fragments, variants or derivatives thereof. Such nucleic acid moleculesmay comprise an attP2 nucleotide sequence having the sequence set forthin FIG. 9, such as:CAAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTG-CAACAAATTGATAAGCAATGCTTTCTTATAATGCCAACTTT-GTACAAGAAAGCTGAACGAGAAACGTAAAATGATA-TAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAG-ACTACATAATACTGTAAAACACAACATATCCAGTCACTATGAATCAA-CTACTTAGATGGTATTAGTGACCTGTA(SEQ ID NO:4), or a nucleotide sequence complementary to the nucleotidesequence set forth in FIG. 9 for attP2, or mutants, fragments, variantsor derivatives thereof. As noted above for attB1, certain mutations,insertions, or deletions of one or more bases in the attP2 sequencecontained in the nucleic acid molecules of the invention may be madewithout compromising the structural and functional integrity of thesemolecules; hence, nucleic acid molecules comprising such mutations,insertions, or deletions in the attP2 sequence are encompassed withinthe scope of the invention.

A recombinant host cell comprising a nucleic acid molecule (the attPvector pDONR201, also known as pENTR21-attPkan or pAttPkan; see FIG. 49)containing attP1 and attP2 sites, E. coli DB3.1(pAttPkan) (also calledE. coli DB3.1(pAHKan)), was deposited on Feb. 27, 1999, with theCollection, Agricultural Research Culture Collection (NRRL), 1815 NorthUniversity Street, Peoria, Ill. 61604 USA, as Deposit No. NRRL B-30099.The attP1 and attP2 sites within the deposited nucleic acid molecule arecontained in nucleic acid cassettes in association with one or moreadditional functional sequences as described in more detail below.

In another related aspect, the invention provides nucleic acid moleculescomprising one or more nucleotide sequences encoding attR1, or mutants,fragments, variants or derivatives thereof. Such nucleic acid moleculesmay comprise an attR1 nucleotide sequence having the sequence set forthin FIG. 9, such as:ACAAGTTTGTACAAAAAAGCTGAACGAG-AAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCAT-AAAAAACAGACTACATAATACTGTAAAACACAACATATCCAGTCA-CTATG(SEQ ID NO:5), or a nucleotide sequence complementary to the nucleotidesequence set forth in FIG. 9 for attR1, or mutants, fragments, variantsor derivatives thereof. As noted above for attB1, certain mutations,insertions, or deletions of one or more bases in the attR1 sequencecontained in the nucleic acid molecules of the invention may be madewithout compromising the structural and functional integrity of thesemolecules; hence, nucleic acid molecules comprising such mutations,insertions, or deletions in the attR1 sequence are encompassed withinthe scope of the invention.

In another related aspect, the invention provides nucleic acid moleculescomprising one or more nucleotide sequences encoding attR2, or mutants,fragments, variants or derivatives thereof. Such nucleic acid moleculesmay comprise an attR2 nucleotide sequence having the sequence set forthin FIG. 9, such as:GCAGGTCGACCATAGTGACTGGATAT-GTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGCAAAATCTA-ATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTTCAGCTT-TCTTGTACAAAGTGGT(SEQ ID NO:6), or a nucleotide sequence complementary to the nucleotidesequence set forth in FIG. 9 for attR2, or mutants, fragments, variantsor derivatives thereof. As noted above for attB1, certain mutations,insertions, or deletions of one or more bases in the attR2 sequencecontained in the nucleic acid molecules of the invention may be madewithout compromising the structural and functional integrity of thesemolecules; hence, nucleic acid molecules comprising such mutations,insertions, or deletions in the attR2 sequence are encompassed withinthe scope of the invention.

Recombinant host cell strains containing attR1 sites apposed to cloningsites in reading frame A, reading frame B, and reading frame C, E. coliDB3.1(pEZC15101) (reading frame A; see FIG. 64A), E. coliDB3.1(pEZC15102) (reading frame B; see FIG. 64B), and E. coliDB3.1(pEZC15103) (reading frame C; see FIG. 64C), and containingcorresponding attR2 sites, were deposited on Feb. 27, 1999, with theCollection, Agricultural Research Culture Collection (NRRL), 1815 NorthUniversity Street, Peoria, Ill. 61604 USA, as Deposit Nos. NRRL B-30103,NRRL B-30104, and NRRL B-30105, respectively. The attR1 and attR2 siteswithin the deposited nucleic acid molecules are contained in nucleicacid cassettes in association with one or more additional functionalsequences as described in more detail below.

In another related aspect, the invention provides nucleic acid moleculescomprising one or more nucleotide sequences encoding attL1, or mutants,fragments, variants and derivatives thereof. Such nucleic acid moleculesmay comprise an attL1 nucleotide sequence having the sequence set forthin FIG. 9, such as: CAA ATA ATG ATT TTA TTT TGA CTG ATA GTG ACC TGT TCGTTG CAA CAA ATT GAT AAG CAA TGC TTT TTT ATA ATG CCA ACT TTG TAC AAA AAAGCA GGC T (SEQ ID NO:7), or a nucleotide sequence complementary to thenucleotide sequence set forth in FIG. 9 for attL1, or mutants,fragments, variants or derivatives thereof. As noted above for attB1,certain mutations, insertions, or deletions of one or more bases in theattL1 sequence contained in the nucleic acid molecules of the inventionmay be made without compromising the structural and functional integrityof these molecules; hence, nucleic acid molecules comprising suchmutations, insertions, or deletions in the attL1 sequence areencompassed within the scope of the invention.

In another related aspect, the invention provides nucleic acid moleculescomprising one or more nucleotide sequences encoding attL2, or mutants,fragments, variants and derivatives thereof. Such nucleic acid moleculesmay comprise an attL2 nucleotide sequence having the sequence set forthin FIG. 9, such as: C AAA TAA TGA TTT TAT TTT GAC TGA TAG TGA CCT GTTCGT TGC AAC AAA TTG ATA AGC AAT GCT TTC TTA TAA TGC CAA CTT TGT ACA AGAAAG CTG GGT (SEQ ID NO:8), or a nucleotide sequence complementary to thenucleotide sequence set forth in FIG. 9 for attL2, or mutants,fragments, variants or derivatives thereof. As noted above for attB1,certain mutations, insertions, or deletions of one or more bases in theattL2 sequence contained in the nucleic acid molecules of the inventionmay be made without compromising the structural and functional integrityof these molecules; hence, nucleic acid molecules comprising suchmutations, insertions, or deletions in the attL2 sequence areencompassed within the scope of the invention.

Recombinant host cell strains containing attL1 sites apposed to cloningsites in reading frame A, reading frame B, and reading frame C, E. coliDB3.1(pENTR1A) (reading frame A; see FIG. 10), E. coli DB3.1(pENTR2B)(reading frame B; see FIG. 11), and E. coli DB3.1 (pENTR3C) (readingframe C; see FIG. 12), and containing corresponding attL2 sites, weredeposited on Feb. 27, 1999, with the Collection, Agricultural ResearchCulture Collection (NRRL), 1815 North University Street, Peoria, Ill.61604 USA, as Deposit Nos. NRRL B-30100, NRRL B-30101, and NRRL B-30102,respectively. The attL1 and attL2 sites within the deposited nucleicacid molecules are contained in nucleic acid cassettes in associationwith one or more additional functional sequences as described in moredetail below.

Each of the recombination site sequences described herein or portionsthereof, or the nucleotide sequence cassettes contained in the depositedclones, may be cloned or inserted into a vector of interest (forexample, using the recombinational cloning methods described hereinand/or standard restriction cloning techniques that are routine in theart) to generate, for example, Entry Vectors or Destination Vectorswhich may be used to transfer a desired segment of a nucleic acidmolecule of interest (e.g., a gene, cDNA molecule, or cDNA library) intoa desired vector or into a host cell.

Using the information provided herein, such as the nucleotide sequencesfor the recombination site sequences described herein, an isolatednucleic acid molecule of the present invention encoding one or morerecombination sites or portions thereof may be obtained using standardcloning and screening procedures, such as those for cloning cDNAs usingmRNA as starting material. Preferred such methods include PCR-basedcloning methods, such as reverse transcriptase-PCR (RT-PCR) usingprimers such as those described herein and in the Examples below.Alternatively, vectors comprising the cassettes containing therecombination site sequences described herein are available commerciallyfrom Life Technologies, Inc. (Rockville, Md.).

The invention is also directed to nucleic acid molecules comprising oneor more of the recombination site sequences or portions thereof and oneor more additional nucleotide sequences, which may encode functional orstructural sites such as one or more multiple cloning sites, one or moretranscription termination sites, one or more transcriptional regulatorysequences (which may be promoters, enhancers, repressors, and the like),one or more translational signals (e.g., secretion signal sequences),one or more origins of replication, one or more fusion partner peptides(particularly glutathione S-transferase (GST), hexahistidine (His₆), andthioredoxin (Trx)), one or more selection markers or modules, one ormore nucleotide sequences encoding localization signals such as nuclearlocalization signals or secretion signals, one or more origins ofreplication, one or more protease cleavage sites, one or more genes orportions of genes encoding a protein or polypeptide of interest, and oneor more 5′ polynucleotide extensions (particularly an extension ofguanine residues ranging in length from about 1 to about 20, from about2 to about 15, from about 3 to about 10, from about 4 to about 10, andmost preferably an extension of 4 or 5 guanine residues at the 5′ end ofthe recombination site nucleotide sequence. The one or more additionalfunctional or structural sequences may or may not flank one or more ofthe recombination site sequences contained on the nucleic acid moleculesof the invention.

In some nucleic acid molecules of the invention, the one or morenucleotide sequences encoding one or more additional functional orstructural sites may be operably linked to the nucleotide sequenceencoding the recombination site. For example, certain nucleic acidmolecules of the invention may have a promoter sequence operably linkedto a nucleotide sequence encoding a recombination site or portionthereof of the invention, such as a T7 promoter, a phage lambda PLpromoter, an E. coli lac, tip or tac promoter, and other suitablepromoters which will be familiar to the skilled artisan.

Nucleic acid molecules of the present invention, which may be isolatednucleic acid molecules, may be in the form of RNA, such as mRNA, or inthe form of DNA, including, for instance, cDNA and genomic DNA obtainedby cloning or produced synthetically, or in the form of DNA-RNA hybrids.The nucleic acid molecules of the invention may be double-stranded orsingle-stranded. Single-stranded DNA or RNA may be the coding strand,also known as the sense strand, or it may be the non-coding strand, alsoreferred to as the anti-sense strand. The nucleic acid molecules of theinvention may also have a number of topologies, including linear,circular, coiled, or supercoiled.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained in avector are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells, andthose DNA molecules purified (partially or substantially) from asolution whether produced by recombinant DNA or synthetic chemistrytechniques. Isolated RNA molecules include in vivo or in vitro RNAtranscripts of the DNA molecules of the present invention.

The present invention further relates to mutants, fragments, variantsand derivatives of the nucleic acid molecules of the present invention,which encode portions, analogs or derivatives of one or morerecombination sites. Variants may occur naturally, such as a naturalallelic variant. By an “allelic variant” is intended one of severalalternate forms of a gene occupying a given locus on a chromosome of anorganism (see Lewin, B., ed., Genes II, John Wiley & Sons, New York(1985)). Non-naturally occurring variants may be produced usingart-known mutagenesis techniques, such as those described hereinbelow.

Such variants include those produced by nucleotide substitutions,deletions or additions or portions thereof, or combinations thereof. Thesubstitutions, deletions or additions may involve one or morenucleotides. The variants may be altered in coding regions, non-codingregions, or both. Alterations in the coding regions may produceconservative or non-conservative amino acid substitutions, deletions oradditions. Especially preferred among these are silent substitutions,additions and deletions, which do not alter the properties andactivities of the encoded polypeptide(s) or portions thereof, and whichalso do not substantially alter the reactivities of the recombinationsite nucleic acid sequences in recombination reactions. Also especiallypreferred in this regard are conservative substitutions.

Particularly preferred mutants, fragments, variants, and derivatives ofthe nucleic acid molecules of the invention include, but are not limitedto, insertions, deletions or substitutions of one or more nucleotidebases within the 15 bp core region (GCTTTTTTATACTAA) (SEQ ID NO:9) whichis identical in all four wildtype lambda att sites, attB, attP, attL andattR (see U.S. application Ser. No. 08/663,002, filed Jun. 7, 1996 (nowU.S. Pat. No. 5,888,732), Ser. No. 09/005,476, filed Jan. 12, 1998, andSer. No. 09/177,387, filed Oct. 23, 1998, which describes the coreregion in further detail, and the disclosures of which are incorporatedherein by reference in their entireties). Analogously, the core regionsin attB1, attP1, attL1 and attR1 are identical to one another, as arethe core regions in attB2, attP2, attL2 and attR2. Particularlypreferred in this regard are nucleic acid molecules comprisinginsertions, deletions or substitutions of one or more nucleotides withinthe seven by overlap region (TTTATAC, which is defined by the cut sitesfor the integrase protein and is the region where strand exchange takesplace) that occurs within this 15 bp core region (GCTTTTTTATACTAA) (SEQID NO:9). Examples of such preferred mutants, fragments, variants andderivatives according to this aspect of the invention include, but arenot limited to, nucleic acid molecules in which the thymine at position1 of the seven by overlap region has been deleted or substituted with aguanine, cytosine, or adenine; in which the thymine at position 2 of theseven by overlap region has been deleted or substituted with a guanine,cytosine, or adenine; in which the thymine at position 3 of the seven byoverlap region has been deleted or substituted with a guanine, cytosine,or adenine; in which the adenine at position 4 of the seven by overlapregion has been deleted or substituted with a guanine, cytosine, orthymine; in which the thymine at position 5 of the seven by overlapregion has been deleted or substituted with a guanine, cytosine, oradenine; in which the adenine at position 6 of the seven by overlapregion has been deleted or substituted with a guanine, cytosine, orthymine; and in which the cytosine at position 7 of the seven by overlapregion has been deleted or substituted with a guanine, thymine, oradenine; or any combination of one or more such deletions and/orsubstitutions within this seven by overlap region. As described indetail in Example 21 herein, mutants of the nucleic acid molecules ofthe invention in which substitutions have been made within the firstthree positions of the seven by overlap (TTTATAC) have been found in thepresent invention to strongly affect the specificity of recombination,mutant nucleic acid molecules in which substitutions have been made inthe last four positions (TTTATAC) only partially alter recombinationspecificity, and mutant nucleic acid molecules comprising nucleotidesubstitutions outside of the seven by overlap, but elsewhere within the15 bp core region, do not affect specificity of recombination but doinfluence the efficiency of recombination. Hence, in an additionalaspect, the present invention is also directed to nucleic acid moleculescomprising one or more recombination site nucleotide sequences thataffect recombination specificity, particularly one or more nucleotidesequences that may correspond substantially to the seven base pairoverlap within the 15 bp core region, having one or more mutations thataffect recombination specificity. Particularly preferred such moleculesmay comprise a consensus sequence (described in detail in Example 21herein) such as NNNATAC, wherein “N” refers to any nucleotide (i.e., maybe A, G, T/U or C), with the proviso that if one of the first threenucleotides in the consensus sequence is a T/U, then at least one of theother two of the first three nucleotides is not a T/U.

In a related aspect, the present invention is also directed to nucleicacid molecules comprising one or more recombination site nucleotidesequences that enhance recombination efficiency, particularly one ormore nucleotide sequences that may correspond substantially to the coreregion and having one or more mutations that enhance recombinationefficiency. By sequences or mutations that “enhance recombinationefficiency” is meant a sequence or mutation in a recombination site,preferably in the core region (e.g., the 15 bp core region of attrecombination sites), that results in an increase in cloning efficiency(typically measured by determining successful cloning of a testsequence, e.g., by determining CFU/ml for a given cloning mixture) whenrecombining molecules comprising the mutated sequence or core region ascompared to molecules that do not comprise the mutated sequence or coreregion (e.g., those comprising a wildtype recombination site core regionsequence). More specifically, whether or not a given sequence ormutation enhances recombination efficiency may be determined using thesequence or mutation in recombinational cloning as described herein, anddetermining whether the sequence or mutation provides enhancedrecombinational cloning efficiency when compared to a non-mutated (e.g.,wildtype) sequence. Methods of determining preferred cloningefficiency-enhancing mutations for a number of recombination sites,particularly for att recombination sites, are described herein, forexample in Examples 22-25. Examples of preferred such mutantrecombination sites include but are not limited to the attL consensuscore sequence of caacttnntnnnannaagttg (SEQ ID NO:92) (wherein “n”represents any nucleotide), for example the attL5 sequenceagcctgctttattatactaagttggcatta (SEQ ID NO:10) and the attL6 sequenceagcctgcttttttatattaagttggcatta (SEQ ID NO:11); the attB1.6 sequenceggggacaactttgtacaaaaaagttggct (SEQ ID NO:12); the attB2.2 sequenceggggacaactttgtacaagaaagctgggt (SEQ ID NO:13); and the attB2.10 sequenceggggacaactttgtacaagaaagttgggt (SEQ ID NO:14). Those of skill in the artwill appreciate that, in addition to the core region, other portions ofthe att site may affect the efficiency of recombination. There are fiveso-called arm binding sites for the integrase protein in thebacteriophage lambda attP site, two in attR (P1 and P2), and three inattL (P′1, P′2 and P′3). Compared to the core binding sites, theintegrase protein binds to arm sites with high affinity and interactswith core and arm sites through two different domains of the protein. Aswith the core binding site a consensus sequence for the arm binding siteconsisting of C/AAGTCACTAT has been inferred from sequence comparison ofthe five arm binding sites and seven non-att sites (Ross and Landy,Proc. Natl. Acad. Sci. USA 79:7724-7728 (1982)). Each arm site has beenmutated and tested for its effect in the excision and integrationreactions (Numrych et al., Nucl. Acids Res. 18:3953 (1990)). Hence,specific sites are utilized in each reaction in different ways, namely,the P1 and P′3 sites are essential for the integration reaction whereasthe other three sites are dispensable to the integration reaction tovarying degrees. Similarly, the P2, P′1 and P′2 sites are most importantfor the excision reaction, whereas P1 and P′3 are completelydispensable. Interestingly, when P2 is mutated the integration reactionoccurs more efficiently than with the wild type attP site. Similarly,when P1 and P′3 are mutated the excision reaction occurs moreefficiently. The stimulatory effect of mutating integrase arm bindingsites can be explained by removing sites that compete or inhibit aspecific recombination pathway or that function in a reaction thatconverts products back to starting substrates. In fact there is evidencefor an XIS-independent LR reaction (Abremski and Gottesman, J. Mol.Biol. 153:67-78 (1981)). Thus, in addition to modifications in the coreregion of the att site, the present invention contemplates the use ofatt sites containing one or more modifications in the integrase arm-typebinding sites. In some preferred embodiments, one or more mutations maybe introduced into one or more of the P1, P′1, P2, P′2 and P′3 sites. Insome preferred embodiments, multiple mutations may be introduced intoone or more of these sites. Preferred such mutations include those whichincrease the recombination in vitro. For example, in some embodimentsmutations may be introduced into the arm-type binding sites such thatintegrative recombination, corresponding to the BP reaction, isenhanced. In other embodiments, mutations may be introduced into thearm-type binding sites such that excisive recombination, correspondingto the LR reaction, is enhanced. Of course, based on the guidancecontained herein, particularly in the construction and evaluation ofeffects of mutated recombination sites upon recombinational specificityand efficiency, analogous mutated or engineered sequences may beproduced for other recombination sites described herein (including butnot limited to lox, FRT, and the like) and used in accordance with theinvention. For example, much like the mutagenesis strategy used toselect core binding sites that enhance recombination efficiency, similarstrategies can be employed to select changes in the arms of attP, attLand attR, and in analogous sequences in other recombination sites suchas lox, FRT and the like, that enhance recombination efficiency. Hence,the construction and evaluation of such mutants is well within theabilities of those of ordinary skill in the art without undueexperimentation. One suitable methodology for preparing and evaluatingsuch mutations is found in Numrych, et al., (1990) Nucleic AcidsResearch 18(13): 3953-3959.

Other mutant sequences and nucleic acid molecules that may be suitableto enhance recombination efficiency will be apparent from thedescription herein, or may be easily determined by one of ordinary skillusing only routine experimentation in molecular biology in view of thedescription herein and information that is readily available in the art

Since the genetic code is well known in the art, it is also routine forone of ordinary skill in the art to produce degenerate variants of thenucleic acid molecules described herein without undue experimentation.Hence, nucleic acid molecules comprising degenerate variants of nucleicacid sequences encoding the recombination sites described herein arealso encompassed within the scope of the invention.

Further embodiments of the invention include isolated nucleic acidmolecules comprising a polynucleotide having a nucleotide sequence atleast 50% identical, at least 60% identical, at least 70% identical, atleast 75% identical, at least 80% identical, at least 85% identical, atleast 90% identical, and more preferably at least 95%, 96%, 97%, 98% or99% identical to the nucleotide sequences of the seven by overlap regionwithin the 15 bp core region of the recombination sites describedherein, or the nucleotide sequences of attB1, attB2, attP1, attP2,attL1, attL2, attR1 or attR2 as set forth in FIG. 9 (or portionsthereof), or a nucleotide sequence complementary to any of thesenucleotide sequences, or fragments, variants, mutants, and derivativesthereof.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding a particularrecombination site or portion thereof is intended that the nucleotidesequence of the polynucleotide is identical to the reference sequenceexcept that the polynucleotide sequence may include up to five pointmutations (e.g., insertions, substitutions, or deletions) per each 100nucleotides of the reference nucleotide sequence encoding therecombination site. For example, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference attB1nucleotide sequence, up to 5% of the nucleotides in the attB1 referencesequence may be deleted or substituted with another nucleotide, or anumber of nucleotides up to 5% of the total nucleotides in the attB1reference sequence may be inserted into the attB1 reference sequence.These mutations of the reference sequence may occur at the 5′ or 3′terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongnucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identical to, for instance, a given recombination site nucleotidesequence or portion thereof can be determined conventionally using knowncomputer programs such as DNAsis software (Hitachi Software, San Bruno,Calif.) for initial sequence alignment followed by ESEE version 3.0DNA/protein sequence software (cabot@trog.mbb.sfu.ca) for multiplesequence alignments. Alternatively, such determinations may beaccomplished using the BESTFIT program (Wisconsin Sequence AnalysisPackage, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711), which employs a local homology algorithm(Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981))to find the best segment of homology between two sequences. When usingDNAsis, ESEE, BESTFIT or any other sequence alignment program todetermine whether a particular sequence is, for instance, 95% identicalto a reference sequence according to the present invention, theparameters are set such that the percentage of identity is calculatedover the full length of the reference nucleotide sequence and that gapsin homology of up to 5% of the total number of nucleotides in thereference sequence are allowed.

The present invention is directed to nucleic acid molecules at least50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identicalto the attB1, attB2, attP1, attP2, attL1, attL2, attR1 or attR2nucleotide sequences as set forth in FIG. 9, or to the nucleotidesequence of the deposited clones, irrespective of whether they encodeparticular functional polypeptides. This is because even where aparticular nucleic acid molecule does not encode a particular functionalpolypeptide, one of skill in the art would still know how to use thenucleic acid molecule, for instance, as a hybridization probe or apolymerase chain reaction (PCR) primer.

Mutations can also be introduced into the recombination site nucleotidesequences for enhancing site specific recombination or altering thespecificities of the reactants, etc. Such mutations include, but are notlimited to: recombination sites without translation stop codons thatallow fusion proteins to be encoded; recombination sites recognized bythe same proteins but differing in base sequence such that they reactlargely or exclusively with their homologous partners allowing multiplereactions to be contemplated; and mutations that prevent hairpinformation of recombination sites. Which particular reactions take placecan be specified by which particular partners are present in thereaction mixture.

There are well known procedures for introducing specific mutations intonucleic acid sequences. A number of these are described in Ausubel, F.M. et al., Current Protocols in Molecular Biology, Wiley Interscience,New York (1989-1996). Mutations can be designed into oligonucleotides,which can be used to modify existing cloned sequences, or inamplification reactions. Random mutagenesis can also be employed ifappropriate selection methods are available to isolate the desiredmutant DNA or RNA. The presence of the desired mutations can beconfirmed by sequencing the nucleic acid by well known methods.

The following non-limiting methods can be used to modify or mutate agiven nucleic acid molecule encoding a particular recombination site toprovide mutated sites that can be used in the present invention:

-   -   1. By recombination of two parental DNA sequences by        site-specific (e.g. attL and attR to give attP) or other (e.g.        homologous) recombination mechanisms where the parental DNA        segments contain one or more base alterations resulting in the        final mutated nucleic acid molecule;    -   2. By mutation or mutagenesis (site-specific, PCR, random,        spontaneous, etc) directly of the desired nucleic acid molecule;    -   3. By mutagenesis (site-specific, PCR, random, spontaneous, etc)        of parental DNA sequences, which are recombined to generate a        desired nucleic acid molecule;    -   4. By reverse transcription of an RNA encoding the desired core        sequence; and    -   5. By de novo synthesis (chemical synthesis) of a sequence        having the desired base changes, or random base changes followed        by sequencing or functional analysis according to methods that        are routine in the art.

The functionality of the mutant recombination sites can be demonstratedin ways that depend on the particular characteristic that is desired.For example, the lack of translation stop codons in a recombination sitecan be demonstrated by expressing the appropriate fusion proteins.Specificity of recombination between homologous partners can bedemonstrated by introducing the appropriate molecules into in vitroreactions, and assaying for recombination products as described hereinor known in the art. Other desired mutations in recombination sitesmight include the presence or absence of restriction sites, translationor transcription start signals, protein binding sites, particular codingsequences, and other known functionalities of nucleic acid basesequences. Genetic selection schemes for particular functionalattributes in the recombination sites can be used according to knownmethod steps. For example, the modification of sites to provide (from apair of sites that do not interact) partners that do interact could beachieved by requiring deletion, via recombination between the sites, ofa DNA sequence encoding a toxic substance. Similarly, selection forsites that remove translation stop sequences, the presence or absence ofprotein binding sites, etc., can be easily devised by those skilled inthe art.

Accordingly, the present invention also provides a nucleic acidmolecule, comprising at least one DNA segment having at least one, andpreferably at least two, engineered recombination site nucleotidesequences of the invention flanking a selectable marker and/or a desiredDNA segment, wherein at least one of said recombination site nucleotidesequences has at least one engineered mutation that enhancesrecombination in vitro in the formation of a Cointegrate DNA or aProduct DNA. Such engineered mutations may be in the core sequence ofthe recombination site nucleotide sequence of the invention; see U.S.application Ser. No. 08/486,139, filed Jun. 7, 1995, Ser. No.08/663,002, filed Jun. 7, 1996 (now U.S. Pat. No. 5,888,732), Ser. No.09/005,476, filed Jan. 12, 1998, and Ser. No. 09/177,387, filed Oct. 23,1998, the disclosures of which are all incorporated herein by referencein their entireties.

While in the preferred embodiment the recombination sites differ insequence and do not interact with each other, it is recognized thatsites comprising the same sequence, which may interact with each other,can be manipulated or engineered to inhibit recombination with eachother. Such conceptions are considered and incorporated herein. Forexample, a protein binding site (e.g., an antibody-binding site, ahistone-binding site, an enzyme-binding site, or a binding site for anynucleic acid molecule-binding protein) can be engineered adjacent to oneof the sites. In the presence of the protein that recognizes theengineered site, the recombinase fails to access the site and anotherrecombination site in the nucleic acid molecule is therefore usedpreferentially. In the cointegrate this site can no longer react sinceit has been changed, e.g., from attB to attL. During or upon resolutionof the cointegrate, the protein can be inactivated (e.g., by antibody,heat or a change of buffer) and the second site can undergorecombination.

The nucleic acid molecules of the invention can have at least onemutation that confers at least one enhancement of said recombination,said enhancement selected from the group consisting of substantially (i)favoring integration; (ii) favoring recombination; (ii) relieving therequirement for host factors; (iii) increasing the efficiency of saidCointegrate DNA or Product DNA formation; (iv) increasing thespecificity of said Cointegrate DNA or Product DNA formation; and (v)adding or deleting protein binding sites.

In other embodiments, the nucleic acid molecules of the invention may bePCR primer molecules, which comprise one or more of the recombinationsite sequences described herein or portions thereof, particularly thoseshown in FIG. 9 (or sequences complementary to those shown in FIG. 9),or mutants, fragments, variants or derivatives thereof, attached at the3′ end to a target-specific template sequence which specificallyinteracts with a target nucleic acid molecule which is to be amplified.Primer molecules according to this aspect of the invention may furthercomprise one or more, (e.g., 1, 2, 3, 4, 5, 10, 20, 25, 50, 100, 500,1000, or more) additional bases at their 5′ ends, and preferablycomprise one or more (particularly four or five) additional bases, whichare preferably guanines, at their 5′ ends, to increase the efficiency ofthe amplification products incorporating the primer molecules in therecombinational cloning system of the invention. Such nucleic acidmolecules and primers are described in detail in the examples herein,particularly in Examples 22-25.

Certain primers of the invention may comprise one or more nucleotidedeletions in the attB1, attB2, attP1, attP2, attL1, attL2, attR1 orattR2 sequences as set forth in FIG. 9. In one such aspect, for example,attB2 primers may be constructed in which one or more of the first fournucleotides at the 5′ end of the attB2 sequence shown in FIG. 9 havebeen deleted. Primers according to this aspect of the invention maytherefore have the sequence:

(attB2(−1)): (SEQ ID NO: 15) CCCAGCTTTCTTGTACAAAGTGGTnnnnnnnnnnnnn...n(attB2(−2)): (SEQ ID NO: 16) CCAGCTTTCTTGTACAAAGTGGTnnnnnnnnnnnnnn...n(attB2(−3)): (SEQ ID NO: 17) CAGCTTTCTTGTACAAAGTGGTnnnnnnnnnnnnnnn...n(attB2(−4)): (SEQ ID NO: 18) AGCTTTCTTGTACAAAGTGGTnnnnnnnnnnnnnnnn...n,wherein “nnnnnnnnnnnnn . . . n” at the 3′ end of the primer represents atarget-specific sequence of any length, for example from one base up toall of the bases of a target nucleic acid molecule (e.g., a gene) or aportion thereof, the sequence and length which will depend upon theidentity of the target nucleic acid molecule which is to be amplified.

The primer nucleic acid molecules according to this aspect of theinvention may be produced synthetically by attaching the recombinationsite sequences depicted in FIG. 9, or portions thereof, to the 5′ end ofa standard PCR target-specific primer according to methods that arewell-known in the art. Alternatively, additional primer nucleic acidmolecules of the invention may be produced synthetically by adding oneor more nucleotide bases, which preferably correspond to one or more,preferably five or more, and more preferably six or more, contiguousnucleotides of the au nucleotide sequences described herein (see, e.g.,Example 20 herein; see also U.S. application Ser. No. 08/663,002, filedJun. 7, 1996 (now U.S. Pat. No. 5,888,732), Ser. No. 09/005,476, filedJan. 12, 1998, and Ser. No. 09/177,387, filed Oct. 23, 1998, thedisclosures of which are all incorporated herein by reference in theirentireties), to the 5′ end of a standard PCR target-specific primeraccording to methods that are well-known in the art, to provide primershaving the specific nucleotide sequences described herein. As notedabove, primer nucleic acid molecules according to this aspect of theinvention may also optionally comprise one, two, three, four, five, ormore additional nucleotide bases at their 5′ ends, and preferably willcomprise four or five guanines at their 5′ ends. In one particularlypreferred such aspect, the primer nucleic acid molecules of theinvention may comprise one or more, preferably five or more, morepreferably six or more, still more preferably 6-18 or 6-25, and mostpreferably 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24 or 25, contiguous nucleotides or by of the attB1 or attB2nucleotide sequences depicted in FIG. 9 (or nucleotides complementarythereto), linked to the 5′ end of a target-specific (e.g., agene-specific) primer molecule. Primer nucleic acid molecules accordingto this aspect of the invention include, but are not limited to, attB1-and attB2-derived primer nucleic acid molecules having the followingnucleotide sequences:

(SEQ ID NO: 19) ACAAGTTTGTACAAAAAAGCAGGCT-nnnnnnnnnnnnn...n(SEQ ID NO: 20) ACCACTTTGTACAAGAAAGCTGGGT-nnnnnnnnnnnnn...n(SEQ ID NO: 21) TGTACAAAAAAGCAGGCT-nnnnnnnnnnnnn...n  (SEQ ID NO: 22)TGTACAAGAAAGCTGGGT-nnnnnnnnnnnnn...n  (SEQ ID NO: 23)ACAAAAAAGCAGGCT-nnnnnnnnnnnnn...n  (SEQ ID NO: 24)ACAAGAAAGCTGGGT-nnnnnnnnnnnnn...n  (SEQ ID NO: 25)AAAAAGCAGGCT-nnnnnnnnnnnnn...n  (SEQ ID NO: 26)AGAAAGCTGGGT-nnnnnnnnnnnnn...n  (SEQ ID NO: 27)AAAAGCAGGCT-nnnnnnnnnnnnn...n  (SEQ ID NO: 28)GAAAGCTGGGT-nnnnnnnnnnnnn...n  (SEQ ID NO: 29)AAAGCAGGCT-nnnnnnnnnnnnn...n  (SEQ ID NO: 30)AAAGCTGGGT-nnnnnnnnnnnnn...n  AAGCAGGCT-nnnnnnnnnnnnn...nAAGCTGGGT-nnnnnnnnnnnnn...n AGCAGGCT-nnnnnnnnnnnnn...nAGCTGGGT-nnnnnnnnnnnnn...n GCAGGCT-nnnnnnnnnnnnn...nGCTGGGT-nnnnnnnnnnnnn...n CAGGCT-nnnnnnnnnnnnn...nCTGGGT-nnnnnnnnnnnnn...n,wherein “nnnnnnnnnnnnn . . . n” at the 3′ end of the primer represents atarget-specific sequence of any length, for example from one base up toall of the bases of a target nucleic acid molecule (e.g., a gene) or aportion thereof, the sequence and length which will depend upon theidentity of the target nucleic acid molecule which is to be amplified.

Of course, it will be apparent to one of ordinary skill from theteachings contained herein that additional primer nucleic acid moleculesanalogous to those specifically described herein may be produced usingone or more, preferably five or more, more preferably six or more, stillmore preferably ten or more, 15 or more, 20 or more, 25 or more, 30 ormore, etc. (through to and including all) of the contiguous nucleotidesor by of the attP1, attP2, attL1, attL2, attR1 or attR2 nucleotidesequences depicted in FIG. 9 (or nucleotides complementary thereto),linked to the 5′ end of a target-specific (e.g., a gene-specific) primermolecule. As noted above, such primer nucleic acid molecules mayoptionally further comprise one, two, three, four, five, or moreadditional nucleotide bases at their 5′ ends, and preferably willcomprise four guanines at their 5′ ends. Other primer moleculescomprising the attB1, attB2, attP1, attP2, attL1, attL2, attR1 and attR2sequences depicted in FIG. 9, or portions thereof, may be made by one ofordinary skill without resorting to undue experimentation in accordancewith the guidance provided herein.

The primers of the invention described herein are useful in producingPCR fragments having a nucleic acid molecule of interest flanked at eachend by a recombination site sequence (as described in detail below inExample 9), for use in cloning of PCR-amplified DNA fragments using therecombination system of the invention (as described in detail below inExamples 8, 19 and 21-25).

Vectors

The invention also relates to vectors comprising one or more of thenucleic acid molecules of the invention, as described herein. Inaccordance with the invention, any vector may be used to construct thevectors of the invention. In particular, vectors known in the art andthose commercially available (and variants or derivatives thereof) mayin accordance with the invention be engineered to include one or morenucleic acid molecules encoding one or more recombination sites (orportions thereof), or mutants, fragments, or derivatives thereof, foruse in the methods of the invention. Such vectors may be obtained from,for example, Vector Laboratories Inc., InVitrogen, Promega, Novagen, NewEngland Biolabs, Clontech, Roche, Pharmacia, EpiCenter, OriGenesTechnologies Inc., Stratagene, Perkin Elmer, Pharmingen, LifeTechnologies, Inc., and Research Genetics. Such vectors may then forexample be used for cloning or subcloning nucleic acid molecules ofinterest. General classes of vectors of particular interest includeprokaryotic and/or eukaryotic cloning vectors, Expression Vectors,fusion vectors, two-hybrid or reverse two-hybrid vectors, shuttlevectors for use in different hosts, mutagenesis vectors, transcriptionvectors, vectors for receiving large inserts and the like.

Other vectors of interest include viral origin vectors (M13 vectors,bacterial phage λ vectors, bacteriophage P1 vectors, adenovirus vectors,herpesvirus vectors, retrovirus vectors, phage display vectors,combinatorial library vectors), high, low, and adjustable copy numbervectors, vectors which have compatible replicons for use in combinationin a single host (pACYC184 and pBR322) and eukaryotic episomalreplication vectors (pCDM8).

Particular vectors of interest include prokaryotic Expression Vectorssuch as pcDNA II, pSL301, pSE280, pSE380, pSE420, pTrcHisA, B, and C,pRSET A, B, and C (Invitrogen, Inc.), pGEMEX-1, and pGEMEX-2 (Promega,Inc.), the pET vectors (Novagen, Inc.), pTrc99A, pKK223-3, the pGEXvectors, pEZZ18, pRIT2T, and pMC1871 (Pharmacia, Inc.), pKK233-2 andpKK388-1 (Clontech, Inc.), and pProEx-HT (Life Technologies, Inc.) andvariants and derivatives thereof. Destination Vectors can also be madefrom eukaryotic Expression Vectors such as pFastBac, pFastBac HT,pFastBac DUAL, pSFV, and pTet-Splice (Life Technologies, Inc.), pEUK-C1,pPUR, pMAM, pMAMneo, pBI101, pBI121, pDR2, pCMVEBNA, and pYACneo(Clontech), pSVK3, pSVL, pMSG, pCH110, and pKK232-8 (Pharmacia, Inc.),p3′SS, pXT1, pSG5, pPbac, pMbac, pMC1neo, and pOG44 (Stratagene, Inc.),and pYES2, pAC360, pBlueBacHis A, B, and C, pVL1392, pBsueBacIII, pCDM8,pcDNA1, pZeoSV, pcDNA3 pREP4, pCEP4, and pEBVHis (Invitrogen, Inc.) andvariants or derivatives thereof.

Other vectors of particular interest include pUC18, pUC19, pBlueScript,pSPORT, cosmids, phagemids, YACs (yeast artificial chromosomes), BACs(bacterial artificial chromosomes), MACs (mammalian artificialchromosomes), pQE70, pQE60, pQE9 (Quiagen), pBS vectors, PhageScriptvectors, BlueScript vectors, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene),pcDNA3 (InVitrogen), pGEX, pTrsfus, pTrc99A, pET-5, pET-9, pKK223-3,pKK233-3, pDR540, pRIT5 (Pharmacia), pSPORT1, pSPORT2, pCMVSPORT2.0 andpSV-SPORT1 (Life Technologies, Inc.) and variants or derivativesthereof.

Additional vectors of interest include pTrxFus, pThioHis, pLEX, pTrcHis,pTrcHis2, pRSET, pBlueBacHis2, pcDNA3.1/His, pcDNA3.1(−)/Myc-His,pSecTag, pEBVHis, pPIC9K, pPIC3.5K, pAO815, pPICZ, pPICZα, pGAPZ,pGAPZα, pBlueBac4.5, pBlueBacHis2, pMelBac, pSinRep5, pSinHis, pIND,pIND(SP1), pVgRXR, pcDNA2.1. pYES2, pZErO1.1, pZErO-2.1, pCR-Blunt,pSE280, pSE380, pSE420, pVL1392, pVL1393, pCDM8, pcDNA1.1, pcDNA1.1/Amp,pcDNA3.1, pcDNA3.1/Zeo, pSe, SV2, pRc/CMV2, pRc/RSV, pREP4, pREP7,pREP8, pREP9, pREP10, pCEP4, pEBVHis, pCR3.1, pCR2.1, pCR3.1-Uni, andpCRBac from Invitrogen; λExCell, λgt11, pTrc99A, pKK223-3, pGEX-1λT,pGEX-2T, pGEX-2TK, pGEX-4T-1, pGEX-4T-2, pGEX-4T-3, pGEX-3X, pGEX-5X-1,pGEX-5X-2, pGEX-5X-3, pEZZ18, pRIT2T, pMC1871, pSVK3, pSVL, pMSG,pCH110, pKK232-8, pSL1180, pNEO, and pUC4K from Pharmacia;pSCREEN-1b(+), pT7Blue(R), pT7Blue-2, pCITE-4abc(+), pOCUS-2, pTAg,pET-32 LIC, pET-30 LIC, pBAC-2cp LIC, pBACgus-2cp LIC, pT7Blue-2 LIC,pT7Blue-2, λSCREEN-1, λBlueSTAR, pET-3abcd, pET-7abc, pET9abcd,pET11abcd, pET12abc, pET-14b, pET-15b, pET-16b, pET-17b-pET-17xb,pET-19b, pET-20b(+), pET-21abcd(+), pET-22b(+), pET-23abcd(+),pET-24abcd(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28abc(+),pET-29abc(+), pET-30abc(+), pET-31b(+), pET-32abc(+), pET-33b(+),pBAC-1, pBACgus-1, pBAC4x-1, pBACgus4x-1, pBAC-3cp, pBACgus-2cp,pBACsurf-1, plg, Signal plg, pYX, Selecta Vecta-Neo, Selecta Vecta-Hyg,and Selecta Vecta-Gpt from Novagen; pLexA, pB42AD, pGBT9, pAS2-1,pGAD424, pACT2, pGAD GL, pGAD GH, pGAD10, pGilda, pEZM3, pEGFP, pEGFP-1,pEGFP-N, pEGFP-C, pEBFP, pGFPuv, pGFP, p6xHis-GFP, pSEAP2-Basic,pSEAP2-Contral, pSEAP2-Promoter, pSEAP2-Enhancer, pβgal-Basic,pβgal-Control, pβgal-Promoter, pβgal-Enhancer, pCMVβ, pTet-Off, pTet-On,pTK-Hyg, pRetro-Off, pRetro-On, pIRES1neo, pIRES1hyg, pLXSN, pLNCX,pLAPSN, pMAMneo, pMAMneo-CAT, pMAMneo-LUC, pPUR, pSV2neo, pYEX 4T-1/2/3,pYEX-S 1, pBacPAK-His, pBacPAK8/9, pAcUW31, BacPAK6, pTrip1Ex, λgt10,λgt11, pWE15, and λTripIEx from Clontech; Lambda ZAP II, pBK-CMV,pBK-RSV, pBluescript II KS+/−, pBluescript II SK+/−, pAD-GALA-, pBD-GAL4Cam, pSurfscript, Lambda FIX II, Lambda DASH, Lambda EMBL3, LambdaEMBL4, SuperCos, pCR-Scrigt Amp, pCR-Script Cam, pCR-Script Direct,pBS+/−, pBC KS+/−, pBC SK+/−, Phagescript, pCAL-n-EK, pCAL-n, pCAL-c,pCAL-kc, pET-3abcd, pET-11abcd, pSPUTK, pESP-1, pCMVLacI, pOPRSVI/MCS,pOPI3 CAT, pXT1, pSG5, pPbac, pMbac, pMC1neo, pMC1neo Poly A, pOG44,pOG45, pFRTβGAL, pNEOβGAL, pRS403, pRS404, pRS405, pRS406, pRS413,pRS414, pRS415, and pRS416 from Stratagene.

Two-hybrid and reverse two-hybrid vectors of particular interest includepPC86, pDBLeu, pDBTrp, pPC97, p2.5, pGAD1-3, pGAD10, pACt, pACT2,pGADGL, pGADGH, pAS2-1, pGAD424, pGBT8, pGBT9, pGAD-GAL4, pLexA,pBD-GAL4, pHISi, pHISi-1, placZi, pB42AD, pDG202, pJK202, pJG4-5,pNLexA, pYESTrp and variants or derivatives thereof.

Yeast Expression Vectors of particular interest include pESP-1, pESP-2,pESC-His, pESC-Trp, pESC-URA, pESC-Leu (Stratagene), pRS401, pRS402,pRS411, pRS412, pRS421, pRS422, and variants or derivatives thereof.

According to the invention, the vectors comprising one or more nucleicacid molecules encoding one or more recombination sites, or mutants,variants, fragments, or derivatives thereof, may be produced by one ofordinary skill in the art without resorting to undue experimentationusing standard molecular biology methods. For example, the vectors ofthe invention may be produced by introducing one or more of the nucleicacid molecules encoding one or more recombination sites (or mutants,fragments, variants or derivatives thereof) into one or more of thevectors described herein, according to the methods described, forexample, in Maniatis et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982). In arelated aspect of the invention, the vectors may be engineered tocontain, in addition to one or more nucleic acid molecules encoding oneor more recombination sites (or portions thereof), one or moreadditional physical or functional nucleotide sequences, such as thoseencoding one or more multiple cloning sites, one or more transcriptiontermination sites, one or more transcriptional regulatory sequences(e.g., one or more promoters, enhancers, or repressors), one or moreselection markers or modules, one or more genes or portions of genesencoding a protein or polypeptide of interest, one or more translationalsignal sequences, one or more nucleotide sequences encoding a fusionpartner protein or peptide (e.g., GST, His₆ or thioredoxin), one or moreorigins of replication, and one or more 5′ or 3′ polynucleotide tails(particularly a poly-G tail). According to this aspect of the invention,the one or more recombination site nucleotide sequences (or portionsthereof) may optionally be operably linked to the one or more additionalphysical or functional nucleotide sequences described herein.

Preferred vectors according to this aspect of the invention include, butare not limited to: pENTR1A (FIGS. 10A and 10B), pENTR2B (FIGS. 11A and11B), pENTR3C (FIGS. 12A and 12B), pENTR4 (FIGS. 13A and 13B), pENTR5(FIGS. 14A and 14B), pENTR6 (FIGS. 15A and 15B), pENTR7 (FIGS. 16A and16B), pENTR8 (FIGS. 17A and 17B), pENTR9 (FIGS. 18A and 18B), pENTR10(FIGS. 19A and 19B), pENTR11 (FIGS. 20A and 20B), pDEST1 (FIGS. 21A-D),pDEST2 (FIG. 22A-D), pDEST3 (FIG. 23A-D), pDEST4 (FIG. 24A-D), pDEST5(FIG. 25A-D), pDEST6 (FIG. 26A-D), pDEST7 (FIG. 27A-C), pDEST8 (FIG.28A-D), pDEST9 (FIG. 29A-E), pDEST10 (FIG. 30A-D), pDEST11 (FIG. 31A-D),pDEST12.2 (also known as pDEST12) (FIG. 32A-D), pDEST13 (FIG. 33A-C),pDEST14 (FIG. 34A-D), pDEST15 (FIG. 35A-D), pDEST16 (FIG. 36A-D),pDEST17 (FIG. 37A-D), pDEST18 (FIG. 38A-D), pDEST19 (FIG. 39A-D),pDEST20 (FIG. 40A-D), pDEST21 (FIG. 41A-E), pDEST22 (FIG. 42A-D),pDEST23 (FIG. 43A-D), pDEST24 (FIG. 44A-D), pDEST25 (FIG. 45A-D),pDEST26 (FIG. 46A-D), pDEST27 (FIG. 47A-D), pEXP501 (also known aspCMVSPORT6) (FIG. 48A-B), pDONR201 (also known as pENTR21 attP vector orpAttPkan Donor Vector) (FIG. 49), pDONR202 (FIG. 50), pDONR203 (alsoknown as pEZ15812) (FIG. 51), pDONR204 (FIG. 52), pDONR205 (FIG. 53),pDONR206 (also known as pENTR22 attP vector or pAttPgen Donor Vector)(FIG. 54), pMAB58 (FIG. 87), pMAB62 (FIG. 88), pDEST28 (FIG. 90),pDEST29 (FIG. 91), pDEST30 (FIG. 92), pDEST31 (FIG. 93), pDEST32 (FIG.94), pDEST33 (FIG. 95), pDEST34 (FIG. 96), pDONR207 (FIG. 97), pMAB85(FIG. 98), pMAB86 (FIG. 99), and fragments, mutants, variants, andderivatives thereof. However, it will be understood by one of ordinaryskill that the present invention also encompasses other vectors notspecifically designated herein, which comprise one or more of theisolated nucleic acid molecules of the invention encoding one or morerecombination sites or portions thereof (or mutants, fragments, variantsor derivatives thereof), and which may further comprise one or moreadditional physical or functional nucleotide sequences described hereinwhich may optionally be operably linked to the one or more nucleic acidmolecules encoding one or more recombination sites or portions thereof.Such additional vectors may be produced by one of ordinary skillaccording to the guidance provided in the present specification.

Polymerases

Preferred polypeptides having reverse transcriptase activity (i.e.,those polypeptides able to catalyze the synthesis of a DNA molecule froman RNA template) for use in accordance with the present inventioninclude, but are not limited to Moloney Murine Leukemia Virus (M-MLV)reverse transcriptase, Rous Sarcoma Virus (RSV) reverse transcriptase,Avian Myeloblastosis Virus (AMV) reverse transcriptase, Rous AssociatedVirus (RAV) reverse transcriptase, Myeloblastosis Associated Virus (MAV)reverse transcriptase, Human Immunodeficiency Virus (HIV) reversetranscriptase, retroviral reverse transcriptase, retrotransposon reversetranscriptase, hepatitis B reverse transcriptase, cauliflower mosaicvirus reverse transcriptase and bacterial reverse transcriptase.Particularly preferred are those polypeptides having reversetranscriptase activity that are also substantially reduced in RNAse Hactivity (i.e., “RNAse H⁻” polypeptides). By a polypeptide that is“substantially reduced in RNase H activity” is meant that thepolypeptide has less than about 20%, more preferably less than about15%, 10% or 5%, and most preferably less than about 2%, of the RNase Hactivity of a wildtype or RNase H⁺ enzyme such as wildtype M-MLV reversetranscriptase. The RNase H activity may be determined by a variety ofassays, such as those described, for example, in U.S. Pat. No.5,244,797, in Kotewicz, M. L. et al., Nucl. Acids Res. 16:265 (1988) andin Gerard, G. F., et al., FOCUS 14(5):91 (1992), the disclosures of allof which are fully incorporated herein by reference. Suitable RNAse H⁻polypeptides for use in the present invention include, but are notlimited to, M-MLV H⁻ reverse transcriptase, RSV H⁻ reversetranscriptase, AMV H⁻ reverse transcriptase, RAV H⁻ reversetranscriptase, MAV H⁻ reverse transcriptase, HIV H⁻ reversetranscriptase, THERMOSCRIPT™ reverse transcriptase and THERMOSCRIPT ™ IIreverse transcriptase, and SUPERSCRIPT™ I reverse transcriptase andSUPERSCRIPT™ IIreverse transcriptase, which are obtainable, for example,from Life Technologies, Inc. (Rockville, Md.). See generally publishedPCT application WO 98/47912.

Other polypeptides having nucleic acid polymerase activity suitable foruse in the present methods include thermophilic DNA polymerases such asDNA polymerase I, DNA polymerase III, Klenow fragment, T7 polymerase,and T5 polymerase, and thermostable DNA polymerases including, but notlimited to, Thermus thermophilus (Tth) DNA polymerase, Thermus aquaticus(Taq) DNA polymerase, Thermotoga neopolitana (Tne) DNA polymerase,Thermotoga maritima (Tma) DNA polymerase, Thermococcus litoralis (Tli orVENT®) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase,Pyrococcus species GB-D (or DEEPVENT®) DNA polymerase, Pyrococcus woosii(Pwo) DNA polymerase, Bacillus sterothermophilus (Bst) DNA polymerase,Sulfolobus acidocaldarius (Sac) DNA polymerase, Thermoplasma acidophilum(Tac) DNA polymerase, Thermus flavus (Tfl/Tub) DNA polymerase, Thermusruber (Tru) DNA polymerase, Thermus brockianus (DYNAZYME®) DNApolymerase, Methanobacterium thermoautotrophicum (Mth) DNA polymerase,and mutants, variants and derivatives thereof. Such polypeptides areavailable commercially, for example from Life Technologies, Inc.(Rockville, Md.), New Englan BioLabs (Beverly, Mass.), and Sigma/Aldrich(St. Louis, Mo.).

Host Cells

The invention also relates to host cells comprising one or more of thenucleic acid molecules or vectors of the invention, particularly thosenucleic acid molecules and vectors described in detail herein.Representative host cells that may be used according to this aspect ofthe invention include, but are not limited to, bacterial cells, yeastcells, plant cells and animal cells. Preferred bacterial host cellsinclude Escherichia spp. cells (particularly E. coli cells and mostparticularly E. coli strains DH10B, Stb12, DH5α, DB3, DB3.1 (preferablyE. coli LIBRARY EFFICIENCY® DB3.1™ Competent Cells; Life Technologies,Inc., Rockville, Md.), DB4 and DB5; see U.S. Provisional Application No.60/122,392, filed on Mar. 2, 1999, the disclosure of which isincorporated by reference herein in its entirety), Bacillus spp. cells(particularly B. subtilis and B. megaterium cells), Streptomyces spp.cells, Erwinia spp. cells, Klebsiella spp. cells, Serratia spp. cells(particularly S. marcessans cells), Pseudomonas spp. cells (particularlyP. aeruginosa cells), and Salmonella spp. cells (particularly S.typhimurium and S. typhi cells). Preferred animal host cells includeinsect cells (most particularly Drosophila melanogaster cells,Spodoptera frugiperda Sf9 and Sf21 cells and Trichoplusa High-Fivecells), nematode cells (particularly C. elegans cells), avian cells,amphibian cells (particularly Xenopus laevis cells), reptilian cells,and mammalian cells (most particularly CHO, COS, VERO, BILK and humancells). Preferred yeast host cells include Saccharomyces cerevisiaecells and Pichia pastoris cells. These and other suitable host cells areavailable commercially, for example from Life Technologies, Inc.(Rockville, Md.), American Type Culture Collection (Manassas, Va.), andAgricultural Research Culture Collection (NRRL; Peoria, Ill.).

Methods for introducing the nucleic acid molecules and/or vectors of theinvention into the host cells described herein, to produce host cellscomprising one or more of the nucleic acid molecules and/or vectors ofthe invention, will be familiar to those of ordinary skill in the art.For instance, the nucleic acid molecules and/or vectors of the inventionmay be introduced into host cells using well known techniques ofinfection, transduction, transfection, and transformation. The nucleicacid molecules and/or vectors of the invention may be introduced aloneor in conjunction with other the nucleic acid molecules and/or vectors.Alternatively, the nucleic acid molecules and/or vectors of theinvention may be introduced into host cells as a precipitate, such as acalcium phosphate precipitate, or in a complex with a lipid.Electroporation also may be used to introduce the nucleic acid moleculesand/or vectors of the invention into a host. Likewise, such moleculesmay be introduced into chemically competent cells such as E. coli. Ifthe vector is a virus, it may be packaged in vitro or introduced into apackaging cell and the packaged virus may be transduced into cells.Hence, a wide variety of techniques suitable for introducing the nucleicacid molecules and/or vectors of the invention into cells in accordancewith this aspect of the invention are well known and routine to those ofskill in the art. Such techniques are reviewed at length, for example,in Sambrook, J., et al., Molecular Cloning, a Laboratory Manual, 2ndEd., Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, pp.16.30-16.55 (1989), Watson, J. D., et al., Recombinant DNA, 2nd Ed., NewYork: W.H. Freeman and Co., pp. 213-234 (1992), and Winnacker, E.-L.,From Genes to Clones, New York: VCH Publishers (1987), which areillustrative of the many laboratory manuals that detail these techniquesand which are incorporated by reference herein in their entireties fortheir relevant disclosures.

Polypeptides

In another aspect, the invention relates to polypeptides encoded by thenucleic acid molecules of the invention (including polypeptides andamino acid sequences encoded by all possible reading frames of thenucleic acid molecules of the invention), and to methods of producingsuch polypeptides. Polypeptides of the present invention includepurified or isolated natural products, products of chemical syntheticprocedures, and products produced by recombinant techniques from aprokaryotic or eukaryotic host, including, for example, bacterial,yeast, insect, mammalian, avian and higher plant cells.

The polypeptides of the invention may be produced by synthetic organicchemistry, and are preferably produced by standard recombinant methods,employing one or more of the host cells of the invention comprising thevectors or isolated nucleic acid molecules of the invention. Accordingto the invention, polypeptides are produced by cultivating the hostcells of the invention (which comprise one or more of the nucleic acidmolecules of the invention, preferably contained within an ExpressionVector) under conditions favoring the expression of the nucleotidesequence contained on the nucleic acid molecule of the invention, suchthat the polypeptide encoded by the nucleic acid molecule of theinvention is produced by the host cell. As used herein, “conditionsfavoring the expression of the nucleotide sequence” or “conditionsfavoring the production of a polypeptide” include optimal physical(e.g., temperature, humidity, etc.) and nutritional (e.g., culturemedium, ionic) conditions required for production of a recombinantpolypeptide by a given host cell. Such optimal conditions for a varietyof host cells, including prokaryotic (bacterial), mammalian, insect,yeast, and plant cells will be familiar to one of ordinary skill in theart, and may be found, for example, in Sambrook, J., et al., MolecularCloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor, N.Y.: ColdSpring Harbor Laboratory Press, (1989), Watson, J. D., et al.,Recombinant DNA, 2nd Ed., New York: W.H. Freeman and Co., and Winnacker,E.-L., From Genes to Clones, New York: VCH Publishers (1987).

In some aspects, it may be desirable to isolate or purify thepolypeptides of the invention (e.g., for production of antibodies asdescribed below), resulting in the production of the polypeptides of theinvention in isolated form. The polypeptides of the invention can berecovered and purified from recombinant cell cultures by well-knownmethods of protein purification that are routine in the art, includingammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxylapatite chromatography and lectin chromatography. For example,His6 or GST fusion tags on polypeptides made by the methods of theinvention may be isolated using appropriate affinity chromatographymatrices which bind polypeptides bearing His6 or GST tags, as will befamiliar to one of ordinary skill in the art. Polypeptides of thepresent invention include naturally purified products, products ofchemical synthetic procedures, and products produced by recombinanttechniques from a prokaryotic or eukaryotic host, including, forexample, bacterial, yeast, higher plant, insect and mammalian cells.Depending upon the host employed in a recombinant production procedure,the polypeptides of the present invention may be glycosylated or may benon-glycosylated. In addition, polypeptides of the invention may alsoinclude an initial modified methionine residue, in some cases as aresult of host-mediated processes.

Isolated polypeptides of the invention include those comprising theamino acid sequences encoded by one or more of the reading frames of thepolynucleotides comprising one or more of the recombinationsite-encoding nucleic acid molecules of the invention, including thoseencoding attB1, attB2, attP1, attP2, attL1, attL2, attR1 and attR2having the nucleotide sequences set forth in FIG. 9 (or nucleotidesequences complementary thereto), or fragments, variants, mutants andderivatives thereof; the complete amino acid sequences encoded by thepolynucleotides contained in the deposited clones described herein; theamino acid sequences encoded by polynucleotides which hybridize understringent hybridization conditions to polynucleotides having thenucleotide sequences encoding the recombination site sequences of theinvention as set forth in FIG. 9 (or a nucleotide sequence complementarythereto); or a peptide or polypeptide comprising a portion or a fragmentof the above polypeptides. The invention also relates to additionalpolypeptides having one or more additional amino acids linked (typicallyby peptidyl bonds to form a nascent polypeptide) to the polypeptidesencoded by the recombination site nucleotide sequences or the depositedclones. Such additional amino acid residues may comprise one or morefunctional peptide sequences, for example one or more fusion partnerpeptides (e.g., GST, His₆, Trx, etc.) and the like.

As used herein, the terms “protein,” “peptide,” “oligopeptide” and“polypeptide” are considered synonymous (as is commonly recognized) andeach term can be used interchangeably as the context requires toindicate a chain of two or more amino acids, preferably five or moreamino acids, or more preferably ten or more amino acids, coupled by (a)peptidyl linkage(s), unless otherwise defined in the specific contextsbelow. As is commonly recognized in the art, all polypeptide formulas orsequences herein are written from left to right and in the directionfrom amino terminus to carboxy terminus.

It will be recognized by those of ordinary skill in the art that someamino acid sequences of the polypeptides of the invention can be variedwithout significant effect on the structure or function of thepolypeptides. If such differences in sequence are contemplated, itshould be remembered that there will be critical areas on the proteinwhich determine structure and activity. In general, it is possible toreplace residues which form the tertiary structure, provided thatresidues performing a similar function are used. In other instances, thetype of residue may be completely unimportant if the alteration occursat a non-critical region of the polypeptide.

Thus, the invention further includes variants of the polypeptides of theinvention, including allelic variants, which show substantial structuralhomology to the polypeptides described herein, or which include specificregions of these polypeptides such as the portions discussed below. Suchmutants may include deletions, insertions, inversions, repeats, and typesubstitutions (for example, substituting one hydrophilic residue foranother, but not strongly hydrophilic for strongly hydrophobic as arule). Small changes or such “neutral” or “conservative” amino acidsubstitutions will generally have little effect on activity.

Typical conservative substitutions are the replacements, one foranother, among the aliphatic amino acids Ala, Val, Leu and Ile;interchange of the hydroxylated residues Ser and Thr; exchange of theacidic residues Asp and Glu; substitution between the amidated residuesAsn and Gln; exchange of the basic residues Lys and Arg; andreplacements among the aromatic residues Phe and Tyr.

Thus, the fragment, derivative or analog of the polypeptides of theinvention, such as those comprising peptides encoded by therecombination site nucleotide sequences described herein, may be (i) onein which one or more of the amino acid residues are substituted with aconservative or non-conservative amino acid residue (preferably aconservative amino acid residue), and such substituted amino acidresidue may be encoded by the genetic code or may be an amino acid(e.g., desmosine, citrulline, ornithine, etc.) that is not encoded bythe genetic code; (ii) one in which one or more of the amino acidresidues includes a substituent group (e.g., a phosphate, hydroxyl,sulfate or other group) in addition to the normal “R” group of the aminoacid; (iii) one in which the mature polypeptide is fused with anothercompound, such as a compound to increase the half-life of thepolypeptide (for example, polyethylene glycol), or (iv) one in whichadditional amino acids are fused to the mature polypeptide, such as animmunoglobulin Fc region peptide, a leader or secretory sequence, asequence which is employed for purification of the mature polypeptide(such as GST) or a proprotein sequence. Such fragments, derivatives andanalogs are intended to be encompassed by the present invention, and arewithin the scope of those skilled in the art from the teachings hereinand the state of the art at the time of invention.

The polypeptides of the present invention are preferably provided in anisolated form, and preferably are substantially purified. Recombinantlyproduced versions of the polypeptides of the invention can besubstantially purified by the one-step method described in Smith andJohnson, Gene 67:31-40 (1988). As used herein, the term “substantiallypurified” means a preparation of an individual polypeptide of theinvention wherein at least 50%, preferably at least 60%, 70%, or 75% andmore preferably at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% (by mass) of contaminating proteins (i.e., those thatare not the individual polypeptides described herein or fragments,variants, mutants or derivatives thereof) have been removed from thepreparation.

The polypeptides of the present invention include those which are atleast about 50% identical, at least 60% identical, at least 65%identical, more preferably at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 96%, at least about 97%, at least about 98% or atleast about 99% identical, to the polypeptides described herein. Forexample, preferred attB1-containing polypeptides of the inventioninclude those that are at least about 50% identical, at least 60%identical, at least 65% identical, more preferably at least about 70%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, at least about 96%, at least about 97%,at least about 98% or at least about 99% identical, to thepolypeptide(s) encoded by the three reading frames of a polynucleotidecomprising a nucleotide sequence of attB1 having a nucleic acid sequenceas set forth in FIG. 9 (or a nucleic acid sequence complementarythereto), to a polypeptide encoded by a polynucleotide contained in thedeposited cDNA clones described herein, or to a polypeptide encoded by apolynucleotide hybridizing under stringent conditions to apolynucleotide comprising a nucleotide sequence of attB1 having anucleic acid sequence as set forth in FIG. 9 (or a nucleic acid sequencecomplementary thereto). Analogous polypeptides may be prepared that areat least about 65% identical, more preferably at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98% or at least about 99% identical, to the attB2, attP1,attP2, attL1, attL2, attR1 and attR2 polypeptides of the invention asdepicted in FIG. 9. The present polypeptides also include portions orfragments of the above-described polypeptides with at least 5, 10, 15,20, or 25 amino acids.

By a polypeptide having an amino acid sequence at least, for example,65% “identical” to a reference amino acid sequence of a givenpolypeptide of the invention is intended that the amino acid sequence ofthe polypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to 35 amino acid alterations pereach 100 amino acids of the reference amino acid sequence of a givenpolypeptide of the invention. In other words, to obtain a polypeptidehaving an amino acid sequence at least 65% identical to a referenceamino acid sequence, up to 35% of the amino acid residues in thereference sequence may be deleted or substituted with another aminoacid, or a number of amino acids up to 35% of the total amino acidresidues in the reference sequence may be inserted into the referencesequence. These alterations of the reference sequence may occur at theamino (N-) or carboxy (C-) terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence. As a practicalmatter, whether a given amino acid sequence is, for example, at least65% identical to the amino acid sequence of a given polypeptide of theinvention can be determined conventionally using known computer programssuch as those described above for nucleic acid sequence identitydeterminations, or more preferably using the CLUSTAL W program(Thompson, J. D., et al., Nucleic Acids Res. 22:4673-4680 (1994)).

The polypeptides of the present invention can be used as molecularweight markers on SDS-PAGE gels or on molecular sieve gel filtrationcolumns using methods well known to those of skill in the art. Inaddition, as described in detail below, the polypeptides of the presentinvention can be used to raise polyclonal and monoclonal antibodieswhich are useful in a variety of assays for detecting proteinexpression, localization, detection of interactions with othermolecules, or for the isolation of a polypeptide (including a fusionpolypeptide) of the invention.

In another aspect, the present invention provides a peptide orpolypeptide comprising an epitope-bearing portion of a polypeptide ofthe invention, which may be used to raise antibodies, particularlymonoclonal antibodies, that bind specifically to a one or more of thepolypeptides of the invention. The epitope of this polypeptide portionis an immunogenic or antigenic epitope of a polypeptide of theinvention. An “immunogenic epitope” is defined as a part of a proteinthat elicits an antibody response when the whole protein is theimmunogen. These immunogenic epitopes are believed to be confined to afew loci on the molecule. On the other hand, a region of a proteinmolecule to which an antibody can bind is defined as an “antigenicepitope.” The number of immunogenic epitopes of a protein generally isless than the number of antigenic epitopes (see, e.g., Geysen et al.,Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983)).

As to the selection of peptides or polypeptides bearing an antigenicepitope (i.e., that contain a region of a protein molecule to which anantibody can bind), it is well-known in the art that relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein (see, e.g., Sutcliffe, J. G., et al., Science219:660-666 (1983)). Peptides capable of eliciting protein-reactive seraare frequently represented in the primary sequence of a protein, can becharacterized by a set of simple chemical rules, and are not confined tothe immunodominant regions of intact proteins (i.e., immunogenicepitopes) or to the amino or carboxy termini. Peptides that areextremely hydrophobic and those of six or fewer residues generally areineffective at inducing antibodies that bind to the mimicked protein;longer peptides, especially those containing proline residues, usuallyare effective (Sutcliffe, J. G., et al., Science 219:660-666 (1983)).

Epitope-bearing peptides and polypeptides of the invention designedaccording to the above guidelines preferably contain a sequence of atleast five, more preferably at least seven or more amino acids containedwithin the amino acid sequence of a polypeptide of the invention.However, peptides or polypeptides comprising a larger portion of anamino acid sequence of a polypeptide of the invention, containing about30 to about 50 amino acids, or any length up to and including the entireamino acid sequence of a given polypeptide of the invention, also areconsidered epitope-bearing peptides or polypeptides of the invention andalso are useful for inducing antibodies that react with the mimickedprotein. Preferably, the amino acid sequence of the epitope-bearingpeptide is selected to provide substantial solubility in aqueoussolvents (i.e., the sequence includes relatively hydrophilic residuesand highly hydrophobic sequences are preferably avoided); sequencescontaining proline residues are particularly preferred.

Non-limiting examples of epitope-bearing polypeptides or peptides thatcan be used to generate antibodies specific for the polypeptides of theinvention include certain epitope-bearing regions of the polypeptidescomprising amino acid sequences encoded by polynucleotides comprisingone or more of the recombination site-encoding nucleic acid molecules ofthe invention, including those encoding attB1, attB2, attP1, attP2,attL1, attL2, attR1 and attR2 having the nucleotide sequences set forthin FIG. 9 (or a nucleotide sequence complementary thereto); the completeamino acid sequences encoded by the three reading frames of thepolynucleotides contained in the deposited clones described herein; andthe amino acid sequences encoded by all reading frames ofpolynucleotides which hybridize under stringent hybridization conditionsto polynucleotides having the nucleotide sequences encoding therecombination site sequences (or portions thereof) of the invention asset forth in FIG. 9 (or a nucleic acid sequence complementary thereto).Other epitope-bearing polypeptides or peptides that may be used togenerate antibodies specific for the polypeptides of the invention willbe apparent to one of ordinary skill in the art based on the primaryamino acid sequences of the polypeptides of the invention describedherein, via the construction of Kyte-Doolittle hydrophilicity andJameson-Wolf antigenic index plots of the polypeptides of the inventionusing, for example, PROTEAN computer software (DNASTAR, Inc.; Madison,Wis.).

The epitope-bearing peptides and polypeptides of the invention may beproduced by any conventional means for making peptides or polypeptidesincluding recombinant means using nucleic acid molecules of theinvention. For instance, a short epitope-bearing amino acid sequence maybe fused to a larger polypeptide which acts as a carrier duringrecombinant production and purification, as well as during immunizationto produce anti-peptide antibodies. Epitope-bearing peptides also may besynthesized using known methods of chemical synthesis (see, e.g., U.S.Pat. No. 4,631,211 and Houghten, R. A., Proc. Natl. Acad. Sci. USA82:5131-5135 (1985), both of which are incorporated by reference hereinin their entireties).

As one of skill in the art will appreciate, the polypeptides of thepresent invention and epitope-bearing fragments thereof may beimmobilized onto a solid support, by techniques that are well-known androutine in the art. By “solid support” is intended any solid support towhich a peptide can be immobilized. Such solid supports include, but arenot limited to nitrocellulose, diazocellulose, glass, polystyrene,polyvinylchloride, polypropylene, polyethylene, dextran, Sepharose,agar, starch, nylon, beads and microtitre plates. Linkage of the peptideof the invention to a solid support can be accomplished by attaching oneor both ends of the peptide to the support. Attachment may also be madeat one or more internal sites in the peptide. Multiple attachments (bothinternal and at the ends of the peptide) may also be used according tothe invention. Attachment can be via an amino acid linkage group such asa primary amino group, a carboxyl group, or a sulfhydryl (SH) group orby chemical linkage groups such as with cyanogen bromide (CNBr) linkagethrough a spacer. For non-covalent attachments to the support, additionof an affinity tag sequence to the peptide can be used such as GST(Smith, D. B., and Johnson, K. S., Gene 67:31 (1988)), polyhistidines(Hochuli, E., et al., J. Chromatog. 411:77 (1987)), or biotin. Suchaffinity tags may be used for the reversible attachment of the peptideto the support. Such immobilized polypeptides or fragments may beuseful, for example, in isolating antibodies directed against one ormore of the polypeptides of the invention, or other proteins or peptidesthat recognize other proteins or peptides that bind to one or more ofthe polypeptides of the invention, as described below.

As one of skill in the art will also appreciate, the polypeptides of thepresent invention and the epitope-bearing fragments thereof describedherein can be combined with one or more fusion partner proteins orpeptides, or portions thereof, including but not limited to GST, His₆,Trx, and portions of the constant domain of immunoglobulins (Ig),resulting in chimeric or fusion polypeptides. These fusion polypeptidesfacilitate purification of the polypeptides of the invention (EP 0 394827; Traunecker et al., Nature 331:84-86 (1988)) for use in analyticalor diagnostic (including high-throughput) format.

Antibodies

In another aspect, the invention relates to antibodies that recognizeand bind to the polypeptides (or epitope-bearing fragments thereof) ornucleic acid molecules (or portions thereof) of the invention. In arelated aspect, the invention relates to antibodies that recognize andbind to one or more polypeptides encoded by all reading frames of one ormore recombination site nucleic acid sequences or portions thereof, orto one or more nucleic acid molecules comprising one or morerecombination site nucleic acid sequences or portions thereof, includingbut not limited to att sites (including attB1, attB2, attP1, attP2,attL1, attL2, attR1, attR2 and the like), lox sites (e.g., loxP,loxP511, and the like), FRT, and the like, or mutants, fragments,variants and derivatives thereof. See generally U.S. Pat. No. 5,888,732,which is incorporated herein by reference in its entirety. Theantibodies of the present invention may be polyclonal or monoclonal, andmay be prepared by any of a variety of methods and in a variety ofspecies according to methods that are well-known in the art. See, forinstance, U.S. Pat. No. 5,587,287; Sutcliffe, J. G., et al., Science2/9:660-666 (1983); Wilson et al., Cell 37: 767 (1984); and Bittle, F.J., et al., J. Gen. Viral. 66:2347-2354 (1985). Antibodies specific forany of the polypeptides or nucleic acid molecules described herein, suchas antibodies specifically binding to one or more of the polypeptidesencoded by the recombination site nucleotide sequences, or one or morenucleic acid molecules, described herein or contained in the depositedclones, antibodies against fusion polypeptides (e.g., binding to fusionpolypeptides between one or more of the fusion partner proteins and oneor more of the recombination site polypeptides of the invention, asdescribed herein), and the like, can be raised against the intactpolypeptides or polynucleotides of the invention or one or moreantigenic polypeptide fragments thereof.

As used herein, the term “antibody” (Ab) may be used interchangeablywith the terms “polyclonal antibody” or “monoclonal antibody” (mAb),except in specific contexts as described below. These terms, as usedherein, are meant to include intact molecules as well as antibodyfragments (such as, for example, Fab and F(ab′)₂ fragments) which arecapable of specifically binding to a polypeptide or nucleic acidmolecule of the invention or a portion thereof. It will therefore beappreciated that, in addition to the intact antibodies of the invention,Fab, F(ab′)₂ and other fragments of the antibodies described herein, andother peptides and peptide fragments that bind one or more polypeptidesor polynucleotides of the invention, are also encompassed within thescope of the invention. Such antibody fragments are typically producedby proteolytic cleavage of intact antibodies, using enzymes such aspapain (to produce Fab fragments) or pepsin (to produce F(ab′)₂fragments). Antibody fragments, and peptides or peptide fragments, mayalso be produced through the application of recombinant DNA technologyor through synthetic chemistry.

Epitope-bearing peptides and polypeptides, and nucleic acid molecules orportions thereof, of the invention may be used to induce antibodiesaccording to methods well known in the art, as generally describedherein (see, e.g., Sutcliffe, et al., supra; Wilson, et al., supra; andBittle, F. J., et al., J. Gen. Virol. 66:2347-2354 (1985)).

Polyclonal antibodies according to this aspect of the invention may bemade by immunizing an animal with one or more of the polypeptides ornucleic acid molecules of the invention described herein or portionsthereof according to standard techniques (see, e.g., Harlow, E., andLane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.:Cold Spring Harbor Laboratory Press (1988); Kaufman, P. B., et al., In:Handbook of Molecular and Cellular Methods in Biology and Medicine, BocaRaton, Fla.: CRC Press, pp. 468-469 (1995)). For producing antibodiesthat recognize and bind to the polypeptides or nucleic acid molecules ofthe invention or portions thereof, animals may be immunized with freepeptide or free nucleic acid molecules; however, antibody titer may beboosted by coupling of the peptide to a macromolecular carrier, such asalbumin, KLH, or tetanus toxoid (particularly for producing antibodiesagainst the nucleic acid molecules of the invention or portions thereof;see Harlow and Lane, supra, at page 154), or to a solid phase carriersuch as a latex or glass microbead. For instance, peptides containingcysteine may be coupled to carrier using a linker such asm-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while otherpeptides may be coupled to carrier using a more general linking agentsuch as glutaraldehyde. Animals such as rabbits, rats and mice may beimmunized with either free (if the polypeptide immunogen is larger thanabout 25 amino acids in length) or carrier-coupled peptides or nucleicacid molecules, for instance, by intraperitoneal and/or intradermalinjection of emulsions containing about 100 μg peptide, polynucleotide,or carrier protein, and Freund's adjuvant. Several booster injectionsmay be needed, for instance, at intervals of about two weeks, to providea useful titer of antibody which can be detected, for example, by ELISAassay using free peptide or nucleic acid molecule adsorbed to a solidsurface. In another approach, cells expressing one or more of thepolypeptides or polynucleotides of the invention or an antigenicfragment thereof can be administered to an animal in order to induce theproduction of sera containing polyclonal antibodies, according toroutine immunological methods. In yet another method, a preparation ofone or more of the polypeptides or polynucleotides of the invention isprepared and purified as described herein, to render it substantiallyfree of natural contaminants. Such a preparation may then be introducedinto an animal in order to produce polyclonal antisera of greaterspecific activity. The titer of antibodies in serum from an immunizedanimal, regardless of the method of immunization used, may be increasedby selection of anti-peptide or anti-polynucleotide antibodies, forinstance, by adsorption to the peptide or polynucleotide on a solidsupport and elution of the selected antibodies according to methods wellknown in the art.

In an alternative method, the antibodies of the present invention aremonoclonal antibodies (or fragments thereof which bind to one or more ofthe polypeptides of the invention). Such monoclonal antibodies can beprepared using hybridoma technology (Kohler et al., Nature 256:495(1975); Köhler et al., Eur. J. Immunol. 6:511 (1976); Köhler et al.,Eur. J. Immunol. 6:292 (1976); Hammerling et al., In: MonoclonalAntibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)).In general, such procedures involve immunizing an animal (preferably amouse) with a polypeptide or polynucleotide of the invention (or afragment thereof), or with a cell expressing a polypeptide orpolynucleotide of the invention (or a fragment thereof). The splenocytesof such mice are extracted and fused with a suitable myeloma cell line.Any suitable myeloma cell line may be employed in accordance with thepresent invention; however, it is preferable to employ the parentmyeloma cell line (SP₂O), available from the American Type CultureCollection, Rockville, Md. After fusion, the resulting hybridoma cellsare selectively maintained in HAT medium, and then cloned by limitingdilution as described by Wands et al. (Gastroenterol. 80:225-232(1981)). The hybridoma cells obtained through such a selection are thenassayed to identify clones which secrete antibodies capable of bindingone or more of the polypeptides or nucleic acid molecules of theinvention, or fragments thereof. Hence, the present invention alsoprovides hybridoma cells and cell lines producing monoclonal antibodiesof the invention, particularly that recognize and bind to one or more ofthe polypeptides or nucleic acid molecules of the invention.

Alternatively, additional antibodies capable of binding to one or moreof the polypeptides of the invention, or fragments thereof, may beproduced in a two-step procedure through the use of anti-idiotypicantibodies. Such a method makes use of the fact that antibodies arethemselves antigens, and that, therefore, it is possible to obtain anantibody which binds to a second antibody. In accordance with thismethod, antibodies specific for one or more of the polypeptides orpolynucleotides of the invention, prepared as described above, are usedto immunize an animal, preferably a mouse. The splenocytes of such ananimal are then used to produce hybridoma cells, and the hybridoma cellsare screened to identify clones which produce an antibody whose abilityto bind to an antibody specific for one or more of the polypeptides orpolynucleotides of the invention can be blocked by polypeptides of theinvention themselves. Such antibodies comprise anti-idiotypic antibodiesto the antibodies recognizing one or more of the polypeptides orpolynucleotides of the invention, and can be used to immunize an animalto induce formation of further antibodies specific for one or more ofthe polypeptides or polynucleotides of the invention.

For use, the antibodies of the invention may optionally be detectablylabeled by covalent or non-covalent attachment of one or more labels,including but not limited to chromogenic, enzymatic, radioisotopic,isotopic, fluorescent, toxic, chemiluminescent, or nuclear magneticresonance contrast agents or other labels.

Examples of suitable enzyme labels include malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast-alcoholdehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphateisomerase, peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholineesterase.

Examples of suitable radioisotopic labels include ³H, ¹¹¹In, ¹²⁵I, ¹³¹I,³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ⁵⁷To, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, ⁹⁰Y, ⁶⁷Cu, ²¹⁷Ci,²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, etc. ¹¹¹In is a preferred isotope where invivo imaging is used since its avoids the problem of dehalogenation ofthe ¹²⁵I or ¹³¹I-labeled monoclonal antibody by the liver. In addition,this radionucleotide has a more favorable gamma emission energy forimaging (Perkins et al., Eur. J. Nucl. Med. 10:296-301 (1985);Carasquillo et al., J. Nucl. Med. 28:281-287 (1987)). For example, ¹¹¹Incoupled to monoclonal antibodies with 1-(P-isothiocyanatobenzyl)-DPTAhas shown little uptake in non-tumorous tissues, particularly the liver,and therefore enhances specificity of tumor localization (Esteban etal., J. Nucl. Med. 28:861-870 (1987)).

Examples of suitable non-radioactive isotopic labels include ¹⁵⁷Gd,⁵⁵Mn, ¹⁶²Dy, ⁵²Tr, and ⁵⁶Fe.

Examples of suitable fluorescent labels include an ¹⁵²Eu label, afluorescein label, an isothiocyanate label, a rhodamine label, aphycoerythrin label, a phycocyanin label, an allophycocyanin label, ano-phthaldehyde label, a green fluorescent protein (GFP) label, and afluorescamine label.

Examples of suitable toxin labels include diphtheria toxin, ricin, andcholera toxin.

Examples of chemiluminescent labels include a luminal label, anisoluminal label, an aromatic acridinium ester label, an imidazolelabel, an acridinium salt label, an oxalate ester label, a luciferinlabel, a luciferase label, and an aequorin label.

Examples of nuclear magnetic resonance contrasting agents include heavymetal nuclei such as Gd, Mn, and iron.

Typical techniques for binding the above-described labels to theantibodies of the invention are provided by Kennedy et al., Clin. Chim.Acta 70:1-31 (1976), and Schurs et al., Clin. Chim. Acta 81:1-40 (1977).Coupling techniques mentioned in the latter are the glutaraldehydemethod, the periodate method, the dimaleimide method, them-maleimidobenzyl-N-hydroxy-succinimide ester method, all of whichmethods are incorporated by reference herein.

It will be appreciated by one of ordinary skill that the antibodies ofthe present invention may alternatively be coupled to a solid support,to facilitate, for example, chromatographic and other immunologicalprocedures using such solid phase-immobilized antibodies. Included amongsuch procedures are the use of the antibodies of the invention toisolate or purify polypeptides comprising one or more epitopes encodedby the nucleic acid molecules of the invention (which may be fusionpolypeptides or other polypeptides of the invention described herein),or to isolate or purify polynucleotides comprising one or morerecombination site sequences of the invention or portions thereof.Methods for isolation and purification of polypeptides (and, by analogy,polynucleotides) by affinity chromatography, for example using theantibodies of the invention coupled to a solid phase support, arewell-known in the art and will be familiar to one of ordinary skill. Theantibodies of the invention may also be used in other applications, forexample to cross-link or couple two or more proteins, polypeptides,polynucleotides, or portions thereof into a structural and/or functionalcomplex. In one such use, an antibody of the invention may have two ormore distinct epitope-binding regions that may bind, for example, afirst polypeptide (which may be a polypeptide of the invention) at oneepitope-binding region on the antibody and a second polypeptide (whichmay be a polypeptide of the invention) at a second epitope-bindingregion on the antibody, thereby bringing the first and secondpolypeptides into close proximity to each other such that the first andsecond polypeptides are able to interact structurally and/orfunctionally (as, for example, linking an enzyme and its substrate tocarry out enzymatic catalysis, or linking an effector molecule and itsreceptor to carry out or induce a specific binding of the effectormolecule to the receptor or a response to the effector molecule mediatedby the receptor). Additional applications for the antibodies of theinvention include, for example, the preparation of large-scale arrays ofthe antibodies, polypeptides, or nucleic acid molecules of theinvention, or portions thereof, on a solid support, for example tofacilitate high-throughput screening of protein or RNA expression byhost cells containing nucleic acid molecules of the invention (known inthe art as “chip array” protocols; see, e.g., U.S. Pat. Nos. 5,856,101,5,837,832, 5,770,456, 5,744,305, 5,631,734, and 5,593,839, which aredirected to production and use of chip arrays of polypeptides (includingantibodies) and polynucleotides, and the disclosures of which areincorporated herein by reference in their entireties). By “solidsupport” is intended any solid support to which an antibody can beimmobilized. Such solid supports include, but are not limited tonitrocellulose, diazocellulose, glass, polystyrene, polyvinylchloride,polycarbonate, polypropylene, polyethylene, dextran, Sepharose, agar,starch, nylon, beads and microtitre plates. Preferred are beads made ofglass, latex or a magnetic material. Linkage of an antibody of theinvention to a solid support can be accomplished by attaching one orboth ends of the antibody to the support. Attachment may also be made atone or more internal sites in the antibody. Multiple attachments (bothinternal and at the ends of the antibody) may also be used according tothe invention. Attachment can be via an amino acid linkage group such asa primary amino group, a carboxyl group, or a sulfhydryl (SH) group orby chemical linkage groups such as with cyanogen bromide (CNBr) linkagethrough a spacer. For non-covalent attachments, addition of an affinitytag sequence to the peptide can be used such as GST (Smith, D. B., andJohnson, K. S., Gene 67:31 (1988)), polyhistidines (Hochuli, E., et al.,J. Chromatog. 411:77 (1987)), or biotin. Alternatively, attachment canbe accomplished using a ligand which binds the Fc region of theantibodies of the invention, e.g., protein A or protein G. Such affinitytags may be used for the reversible attachment of the antibodies to thesupport. Peptides may also be recognized via specific ligand-receptorinteractions or using phage display methodologies that will be familiarto the skilled artisan, for their ability to bind polypeptides of theinvention or fragments thereof.

Kits

In another aspect, the invention provides kits which may be used inproducing the nucleic acid molecules, polypeptides, vectors, host cells,and antibodies, and in the recombinational cloning methods, of theinvention. Kits according to this aspect of the invention may compriseone or more containers, which may contain one or more of the nucleicacid molecules, primers, polypeptides, vectors, host cells, orantibodies of the invention. In particular, a kit of the invention maycomprise one or more components (or combinations thereof) selected fromthe group consisting of one or more recombination proteins (e.g., Int)or auxiliary factors (e.g. IHF and/or Xis) or combinations thereof, oneor more compositions comprising one or more recombination proteins orauxiliary factors or combinations thereof (for example, GATEWAY™ LRClonase™ Enzyme Mix or GATEWAY™ BP Clonase™ Enzyme Mix) one or moreDestination Vector molecules (including those described herein), one ormore Entry Clone or Entry Vector molecules (including those describedherein), one or more primer nucleic acid molecules (particularly thosedescribed herein), one or more host cells (e.g. competent cells, such asE. coli cells, yeast cells, animal cells (including mammalian cells,insect cells, nematode cells, avian cells, fish cells, etc.), plantcells, and most particularly E. coli DB3, DB3.1 (preferably E. coliLIBRARY EFFICIENCY® DB3.1™ Competent Cells; Life Technologies, Inc.,Rockville, Md.), DB4 and DB5; see U.S. Provisional Application No.60/122,392, filed on Mar. 2, 1999, and the corresponding U.S. Utilityapplication Ser. No. 09/518,188 of Hartley et al., entitled “CellsResistant to Toxic Genes and Uses Thereof,” filed on even day herewith,the disclosures of which are incorporated by reference herein in itsentirety), and the like. In related aspects, the kits of the inventionmay comprise one or more nucleic acid molecules encoding one or morerecombination sites or portions thereof, such as one or more nucleicacid molecules comprising a nucleotide sequence encoding the one or morerecombination sites (or portions thereof) of the invention, andparticularly one or more of the nucleic acid molecules contained in thedeposited clones described herein. Kits according to this aspect of theinvention may also comprise one or more isolated nucleic acid moleculesof the invention, one or more vectors of the invention, one or moreprimer nucleic acid molecules of the invention, and/or one or moreantibodies of the invention. The kits of the invention may furthercomprise one or more additional containers containing one or moreadditional components useful in combination with the nucleic acidmolecules, polypeptides, vectors, host cells, or antibodies of theinvention, such as one or more buffers, one or more detergents, one ormore polypeptides having nucleic acid polymerase activity, one or morepolypeptides having reverse transcriptase activity, one or moretransfection reagents, one or more nucleotides, and the like. Such kitsmay be used in any process advantageously using the nucleic acidmolecules, primers, vectors, host cells, polypeptides, antibodies andother compositions of the invention, for example in methods ofsynthesizing nucleic acid molecules (e.g., via amplification such as viaPCR), in methods of cloning nucleic acid molecules (preferably viarecombinational cloning as described herein), and the like.

Optimization of Recombinational Cloning System

The usefulness of a particular nucleic acid molecule, or vectorcomprising a nucleic acid molecule, of the invention in methods ofrecombinational cloning may be determined by any one of a number ofassay methods. For example, Entry and Destination vectors of the presentinvention may be assessed for their ability to function (i.e., tomediate the transfer of a nucleic acid molecule, DNA segment, gene, cDNAmolecule or library from a cloning vector to an Expression Vector) bycarrying out a recombinational cloning reaction as described in moredetail in the Examples below and as described in U.S. application Ser.No. 08/663,002, filed Jun. 7, 1996 (now U.S. Pat. No. 5,888,732), Ser.No. 09/005,476, filed Jan. 12, 1998, Ser. No. 09/177,387, filed Oct. 23,1998, and 60/108,324, filed Nov. 13, 1998, the disclosures of which areincorporated by reference herein in their entireties. Alternatively, thefunctionality of Entry and Destination Vectors prepared according to theinvention may be assessed by examining the ability of these vectors torecombine and create cointegrate molecules, or to transfer a nucleicacid molecule of interest, using an assay such as that described indetail below in Example 19. Analogously, the formulation of compositionscomprising one or more recombination proteins or combinations thereof,for example GATEWAY™ LR Clonase™ Enzyme Mix and GATEWAY™ BP Clonase™Enzyme Mix, may be optimized using assays such as those described belowin Example 18.

Uses

There are a number of applications for the compositions, methods andkits of the present invention. These uses include, but are not limitedto, changing vectors, targeting gene products to intracellularlocations, cleaving fusion tags from desired proteins, operably linkingnucleic acid molecules of interest to regulatory genetic sequences(e.g., promoters, enhancers, and the like), constructing genes forfusion proteins, changing copy number, changing replicons, cloning intophages, and cloning, e.g., PCR products, genomic DNAs, and cDNAs. Inaddition, the nucleic acid molecules, vectors, and host cells of theinvention may be used in the production of polypeptides encoded by thenucleic acid molecules, in the production of antibodies directed againstsuch polypeptides, in recombinational cloning of desired nucleic acidsequences, and in other applications that may be enhanced or facilitatedby the use of the nucleic acid molecules, vectors, and host cells of theinvention.

In particular, the nucleic acid molecules, vectors, host cells,polypeptides, antibodies, and kits of the invention may be used inmethods of transferring one or more desired nucleic acid molecules orDNA segments, for example one or more genes, cDNA molecules or cDNAlibraries, into a cloning or Expression Vector for use in transformingadditional host cells for use in cloning or amplification of, orexpression of the polypeptide encoded by, the desired nucleic acidmolecule or DNA segment. Such recombinational cloning methods which mayadvantageously use the nucleic acid molecules, vectors, and host cellsof the invention, are described in detail in the Examples below, and incommonly owned U.S. application Ser. No. 08/486,139, filed Jun. 7, 1995,Ser. No. 08/663,002, filed Jun. 7, 1996 (now U.S. Pat. No. 5,888,732),Ser. No. 09/005,476, filed Jan. 12, 1998, Ser. No. 09/177,387, filedOct. 23, 1998, and 60/108,324, filed Nov. 13, 1998, the disclosures ofall of which are incorporated by reference herein in their entireties.

It will be understood by one of ordinary skill in the relevant arts thatother suitable modifications and adaptations to the methods andapplications described herein are readily apparent from the descriptionof the invention contained herein in view of information known to theordinarily skilled artisan, and may be made without departing from thescope of the invention or any embodiment thereof. Having now describedthe present invention in detail, the same will be more clearlyunderstood by reference to the following examples, which are includedherewith for purposes of illustration only and are not intended to belimiting of the invention.

EXAMPLES Example 1 Recombination Reactions of Bacteriophage λ

The E. coli bacteriophage λ can grow as a lytic phage, in which case thehost cell is lysed, with the release of progeny virus. Alternatively,lambda can integrate into the genome of its host by a process calledlysogenization (see FIG. 60). In this lysogenic state, the phage genomecan be transmitted to daughter cells for many generations, untilconditions arise that trigger its excision from the genome. At thispoint, the virus enters the lytic part of its life cycle. The control ofthe switch between the lytic and lysogenic pathways is one of the bestunderstood processes in molecular biology (M. Ptashne, A Genetic Switch,Cell Press, 1992).

The integrative and excisive recombination reactions of A, performed invitro, are the basis of Recombinational Cloning System of the presentinvention. They can be represented schematically as follows:

attB×attP

attL×attR (where “×” signifies recombination)

The four att sites contain binding sites for the proteins that mediatethe reactions. The wild type attP, attB, attL, and attR sites containabout 243, 25, 100, and 168 base pairs, respectively. The attB×attPreaction (hereinafter referred to as a “BP Reaction,” or alternativelyand equivalently as an “Entry Reaction” or a “Gateward Reaction”) ismediated by the proteins hit and IHF. The attL×attR reaction(hereinafter referred to as an “LR Reaction,” or alternatively andequivalently as a “Destination Reaction”) is mediated by the proteinshit, IHF, and Xis. Int (integrate) and Xis (excisionase) are encoded bythe A genome, while IHF (integration host factor) is an E. coli protein.For a general review of lambda recombination, see: A. Landy, Ann. Rev.Biochem. 58: 913-949 (1989).

Example 2 Recombination Reactions of the Recombinational Cloning System

The LR Reaction—the exchange of a DNA segment from an Entry Clone to aDestination Vector—is the in vitro version of the A excision reaction:

attL×attR

attB+attP.

There is a practical imperative for this configuration: after an LRReaction in one configuration of the present method, an att site usuallyseparates a functional motif (such as a promoter or a fusion tag) from anucleic acid molecule of interest in an Expression Clone, and the 25 bpattB site is much smaller than the attP, attL, and attR sites.

Note that the recombination reaction is conservative, i.e., there is nonet synthesis or loss of base pairs. The DNA segments that flank therecombination sites are merely switched. The wild type A recombinationsites are modified for purposes of the GATEWAY™ Cloning System, asfollows:

To create certain preferred Destination Vectors, a part (43 bp) of attRwas removed, to make the excisive reaction irreversible and moreefficient (W. Bushman et al., Science 230: 906, 1985). The attR sites inpreferred Destination Vectors of the invention are 125 bp in length.Mutations were made to the core regions of the att sites, for tworeasons: (1) to eliminate stop codons, and (2) to ensure specificity ofthe recombination reactions (i.e., attR1 reacts only with attL1, attR2reacts only with attL2, etc.).

Other mutations were introduced into the short (5 bp) regions flankingthe 15 bp core regions of the attB sites to minimize secondary structureformation in single-stranded forms of attB plasmids, e.g., in phagemidssDNA or in mRNA. Sequences of attB1 and attB2 to the left and right ofa nucleic acid molecule of interest after it has been cloned into aDestination Vector are given in FIG. 6.

FIG. 61 illustrates how an Entry Clone and a Destination Vectorrecombine in the LR Reaction to form a co-integrate, which resolvesthrough a second reaction into two daughter molecules. The two daughtermolecules have the same general structure regardless of which pair ofsites, attL1 and attR1 or attL2 and attR2, react first to form theco-integrate. The segments change partners by these reactions,regardless of whether the parental molecules are both circular, one iscircular and one is linear, or both are linear. In this example,selection for ampicillin resistance carried on the Destination Vector,which also carries the death gene ccdB, provides the means for selectingonly for the desired attB product plasmid.

Example 3 Protein Expression in the Recombinational Cloning System

Proteins are expressed in vivo as a result of two processes,transcription (DNA into RNA), and translation (RNA into protein). For areview of protein expression in prokaryotes and eukaryotes, see Example13 below. Many vectors (pUC, BlueScript, pGem) use interruption of atranscribed lacZ gene for blue-white screening. These plasmids, and manyExpression Vectors, use the lac promoter to control expression of clonedgenes. Transcription from the lac promoter is turned on by adding theinducer IPTG. However, a low level of RNA is made in the absence ofinducer, i.e., the lac promoter is never completely off. The result ofthis “leakiness” is that genes whose expression is harmful to E. colimay prove difficult or impossible to clone in vectors that contain thelac promoter, or they may be cloned only as inactive mutants.

In contrast to other gene expression systems, nucleic acid moleculescloned into an Entry Vector may be designed not to be expressed. Thepresence of the strong transcriptional terminator rrnB (Orosz, et al.,Eur. J. Biochem. 201: 653, 1991) just upstream of the attL1 site keepstranscription from the vector promoters (drug resistance and replicationorigin) from reaching the cloned gene. However, if a toxic gene iscloned into a Destination Vector, the host may be sick, just as in otherexpression systems. But the reliability of subcloning by in vitrorecombination makes it easier to recognize that this has happened—andeasier to try another expression option in accordance with the methodsof the invention, if necessary.

Example 4 Choosing the Right Entry Vector

There are two kinds of choices that must be made in choosing the bestEntry Vector, dictated by (1) the particular DNA segment that is to becloned, and (2) what is to be accomplished with the cloned DNA segment.These factors are critical in the choice of Entry Vector used, becausewhen the desired nucleic acid molecule of interest is moved from theEntry Vector to a Destination Vector, all the base pairs between thenucleic acid molecule of interest and the Int cutting sites in attL1 andattL2 (such as in FIG. 6) move into the Destination Vector as well. Forgenomic DNAs that are not expressed as a result of moving into aDestination Vector, these decisions are not as critical.

For example, if an Entry Vector with certain translation start signalsis used, those sequences will be translated into amino acids if anamino-terminal fusion to the desired nucleic acid molecule of interestis made. Whether the desired nucleic acid molecule of interest is to beexpressed as fusion protein, native protein, or both, dictates whethertranslational start sequences must be included between the attB sites ofthe clone (native protein) or, alternatively, supplied by theDestination Vector (fusion protein). In particular, Entry Clones thatinclude translational start sequences may prove less suitable for makingfusion proteins, as internal initiation of translation at these sitescan decrease the yield of N-terminal fusion protein. These two types ofexpression afforded by the compositions and methods of the invention areillustrated in FIG. 62.

No Entry Vector is likely to be optimal for all applications. Thenucleic acid molecule of interest may be cloned into any of severaloptimal Entry Vectors.

As an example, consider pENTR7 (FIG. 16) and pENTR11 (FIG. 20), whichare useful in a variety of applications, including (but not limited to):

-   -   Cloning cDNAs from most of the commercially available libraries.        The sites to the left and right of the ccdB death gene have been        chosen so that directional cloning is possible if the DNA to be        cloned does not have two or    -   Cloning of genes directionally: SalI, BamHI, XmnI (blunt), or        KpnI on the left of ccdB; NotI, XhoI, XbaI, or EcoRV (blunt), on        the right.    -   Cloning of genes or gene fragments with a blunt amino end at the        XmnI site. The XmnI site has four of the six most favored bases        for eukaryotic expression (see Example 13, below), so that if        the first three bases of the DNA to be cloned are ATG, the open        reading frame (ORF) will be expressed in eukaryotic cells (e.g.,        mammalian cells, insect cells, yeast cells) when it is        transcribed in the appropriate Destination Vector. In addition,        in pENTR11, a Shine-Dalgarno sequence is situated 8 bp upstream,        for initiating protein synthesis in a prokaryotic host cell        (particularly a bacterial cell, such as E. coli) at an ATG.    -   Cleaving off amino terminal fusions (e.g., His₆, GST, or        thioredoxin) using the highly specific TEV (Tobacco Etch Virus)        protease (available from Life Technologies, Inc.). If the        nucleic acid molecule of interest is cloned at the blunt XmnI        site, TEV cleavage will leave two amino acids on the amino end        of the expressed protein.    -   Selecting against uncut or singly cut Entry Vector molecules        during cloning with restriction enzymes and ligase. If the ccdB        gene is not removed with a double digest, it will kill any        recipient E. coli cell that does not contain a mutation that        makes the cell resistant to ccdB (see U.S. Provisional        Application No. 60/122,392, filed on Mar. 2, 1999, the        disclosure of which is incorporated by reference herein in its        entirety).    -   Allowing production of amino fusions with ORFs in all cloning        sites. There are no stop codons (in the attL1 reading frame)        upstream of the ccdB gene.

In addition, pENTR11 is also useful in the following applications:

-   -   Cloning cDNAs that have an NcoI site at the initiating ATG into        the NcoI site. Similar to the XmnI site, this site has four of        the six most favored bases for eukaryotic expression. Also, a        Shine-Dalgarno sequence is situated 8 bp upstream, for        initiating protein synthesis in a prokaryotic host cell        (particularly a bacterial cell, such as E. coli) at an ATG.    -   Producing carboxy fusion proteins with ORFs positioned in phase        with the reading frame convention for carboxy-terminal fusions        (see FIG. 20A).

Table 1 lists some non-limiting examples of Entry Vectors and theircharacteristics, and FIGS. 10-20 show their cloning sites. All of theEntry Vectors listed in Table 1 are available commercially from LifeTechnologies, Inc., Rockville, Md. Other Entry Vectors not specificallylisted here, which comprise alternative or additional features may bemade by one of ordinary skill using routine methods of molecular andcellular biology, in view of the disclosure contained herein.

TABLE 1 Examples of Entry Vectors Native Class of Protein in ProteinMnemonic Entry Distinctive Amino Native Protein Eukaryotic SynthesisDesignation Name Vector Cloning Sites Fusions in E. coli Cells FeaturespENTR- Minimal Alternative Reading frame Good Poor Good Minimal amino1A, 2B, 3C blunt RF Reading A, B, or C; blunt acids between A, B, CFrame cut closest to tag and protein; Vectors attL1 no SD pENTR4 MinimalRestr. Enz. Nco I site Good Poor Good Good Kozac; no Nco Cleavage(common in euk. SD Vectors cDNAs) closest to attL1 pENTR5 Minimal Restr.Enz. Nde I site closest Good Poor Poor at Nde I, No SD; poor NdeCleavage to attL1 Good at Xmn Kozac at Nde, Vectors I good at Xmn pENTR6Minimal Restr. Enz. Sph I site closest Good Poor Poor at Sph I, No SD;poor Sph Cleavage to attL1 Good at Xmn Kozac at Sph, Vectors I good atXmn pENTR7 TEV Blunt TEV Xmn I (blunt) is Good Poor Good at Xmn TEVprotease Cleavage first cloning site I site leaves Gly-Thr Site Presentafter TEV site on amino end of protein; no SD pENTR8 TEV Nco TEV Nco Iis first Good Poor Good TEV protease Cleavage cloning site after leavesGly-Thr Site Present TEV site on amino end of protein; no SD pENTR9 TEVNde TEV Nde I is first Good Poor Poor TEV protease Cleavage cloning siteafter leaves Gly-Thr Site Present TEV site on amino end of protein; noSD, poor Kozac pENTR10 Nde with Good SD for Strong SD; Nde I Poor GoodPoor Strong SD, SD E. coli site, no TEV internal starts in Expressionamino fusions. Poor Kz. No TEV pENTR11 2 X Good SD for Xmn I (blunt)Good Good Good Strong SD/Koz SD + Kozac E. coli and Nco I sites Internalstarts in Expression each preceded by amino fusions. SD and Kozac No TEV

Entry vectors pENTR1A (FIGS. 10A and 10B), pENTR2B (FIGS. 11A and 11B),and pENTR3C (FIGS. 12A and 12B) are almost identical, except that therestriction sites are in different reading frames. Entry vectors pENTR4(FIGS. 13A and 13B), pENTR5 (FIGS. 14A and 14B), and pENTR6 (FIGS. 15Aand 15B) are essentially identical to pENTR1A, except that the bluntDraI site has been replaced with sites containing the ATG methioninecodon: NcoI in pENTR4, NdeI in pENTR5, and SphI in pENTR6. Nucleic acidmolecules that contain one of these sites at the initiating ATG can beconveniently cloned in these Entry vectors. The NcoI site in pENTR4 isespecially useful for expression of nucleic acid molecules in eukaryoticcells, since it contains many of the bases that give efficienttranslation (see Example 13, below). (Nucleic acid molecules of interestcloned into the NdeI site of pENTR5 are not expected to be highlyexpressed in eukaryotic cells, because the cytosine at position −3 fromthe initiating ATG is rare in eukaryotic genes.)

Entry vectors pENTR7 (FIGS. 16A and 16B), pENTR8 (FIGS. 17A and 17B),and pENTR9 (FIGS. 18A and 18B) contain the recognition site for the TEVprotease between the attL1 site and the cloning sites. Cleavage sitesfor XmnI (blunt), NcoI, and NdeI, respectively, are the most 5′ sites inthese Entry vectors. Amino fusions can be removed efficiently if nucleicacid molecules are cloned into these Entry vectors. TEV protease ishighly active and highly specific.

Example 5 Controlling Reading Frame

One of the trickiest tasks in expression of cloned nucleic acidmolecules is making sure the reading frame is correct. (Reading frame isimportant if fusions are being made between two ORFs, for examplebetween a nucleic acid molecule of interest and a His6 or GST domain.)For purposes of the present invention, the following convention has beenadopted: The reading frame of the DNA cloned into any Entry Vector mustbe in phase with that of the attB1 site shown in FIG. 16A, pENTR7.Notice that the six As of the attL1 site are split into two lysinecodons (aaa aaa). The Destination Vectors that make amino fusions wereconstructed such that they enter the attR1 site in this reading frame.Destination Vectors for carboxy terminal fusions were also constructed,including those containing His₆ (pDEST23; FIG. 43), GST (pDEST24; FIG.44), or thioredoxin (pDEST25; FIG. 45) C-terminal fusion sequences.

Therefore, if a nucleic acid molecule of interest is cloned into anEntry Vector so that the aaa aaa reading frame within the attL1 site isin phase with the nucleic acid molecule's ORF, amino terminal fusionswill automatically be correctly phased, for all the fusion tags. This isa significant improvement over the usual case, where each differentvector can have different restriction sites and different readingframes.

See Example 15 for a practical example of how to choose the mostappropriate combinations of Entry Vector and Destination Vector.

Materials

Unless otherwise indicated, the following materials were used in theremaining Examples included herein:

5×LR Reaction Buffer:

-   -   200-250 mM (preferably 250 mM) Tris-HCl, pH 7.5    -   250-350 mM (preferably 320 mM) NaCl    -   1.25-5 mM (preferably 4.75 mM) EDTA    -   12.5-35 mM (preferably 22-35 mM, and most preferably 35 mM)    -   Spermidine-HCl    -   1 mg/ml bovine serum albumin

GATEWAY™ LR Clonase™ Enzyme Mix: per 4 μl of 1×LR Reaction Buffer:

-   -   150 ng carboxy-His6-tagged Int (see U.S. Appl. No. 60/108,324,        filed Nov. 13, 1998, and Ser. No. 09/438,358, filed Nov. 12,        1999, both entirely incorporated by reference herein)    -   25 ng carboxy-His6-tagged Xis (see U.S. Appl. No. 60/108,324,        filed Nov. 13, 1998, and Ser. No. 09/438,358, filed Nov. 12,        1999, both entirely incorporated by reference herein)    -   30 ng IHF    -   50% glycerol

5×BP Reaction Buffer:

-   -   125 mM Tris-HCl, pH 7.5    -   110 mM NaCl    -   25 mM EDTA    -   25 mM Spermidine-HCl    -   5 mg/ml bovine serum albumin

GATEWAY™ BP Clonase™ Enzyme Mix:

Per 4 μl of 1×BP Reaction Buffer:

-   -   200 ng carboxy-His6-tagged Int (see U.S. Appl. No. 60/108,324,        filed Nov. 13, 1998, and Ser. No. 09/438,358, filed Nov. 12,        1999, both entirely incorporated by reference herein)    -   80 ng IHF    -   50% glycerol

10× Clonase Stop Solution:

-   -   50 mM Tris-HCl, pH 8.0    -   1 mM EDTA    -   2 mg/ml Proteinase K

Example 6 LR (“Destination”) Reaction

To create a new Expression Clone containing the nucleic acid molecule ofinterest (and which may be introduced into a host cell, ultimately forproduction of the polypeptide encoded by the nucleic acid molecule), anEntry Clone or Vector containing the nucleic acid molecule of interest,prepared as described herein, is reacted with a Destination Vector. Inthe present example, a β-Gal gene flanked by attL sites is transferredfrom an Entry Clone to a Destination Vector.

Materials needed:

-   -   5×LR Reaction buffer    -   Destination Vector (preferably linearized), 75-150 ng/μl    -   Entry Clone containing nucleic acid molecule of interest,        100-300 ng in 8 μl TE buffer    -   Positive control Entry Clone (pENTR-β-Gal) DNA (See note, below)    -   Positive control Destination Vector, pDEST1 (pTrc), 75 ng/μl    -   GATEWAY™ LR Clonase™ Enzyme Mix (stored at −80° C.)    -   10× Clonase Stop solution    -   pUC19 DNA, 10 pg/μl    -   Chemically competent E. coli cells (competence: ≧1×10⁷ CFU/μg),        400 μl.    -   LB Plates containing ampicillin (100 μg/ml) and methicillin (200        μg/ml)±X-gal and IPTG (See below)

Notes:

Preparation of the Entry Clone DNA: Miniprep DNA that has been treatedwith RNase works well. A reasonably accurate quantitation (±50%) of theDNA to be cloned is advised, as the GATEWAY™ reaction appears to have anoptimum of about 100-300 ng of Entry Clone per 20 μl of reaction mix.

The positive control Entry Clone, pENTR-β-Gal, permits functionalanalysis of clones based on the numbers of expected blue vs. whitecolonies on LB plates containing IPTG+Bluo-gal (or X-gal), in additionto ampicillin (100 μg/ml) and methicillin (200 μg/ml). Becauseβ-Galactosidase is a large protein, it often yields a less prominentband than many smaller proteins do on SDS protein gels.

In the Positive Control Entry Vector pENTR-β-Gal, the coding sequence ofβ-Gal has been cloned into pENTR11 (FIGS. 20A and 20B), withtranslational start signals permitting expression in E. coli, as well asin eukaryotic cells. The positive control Destination Vector, forexample pDEST1 (FIG. 21), is preferably linearized.

To prepare X-gal+IPTG plates, either of the following protocols may beused:

A. With a glass rod, spread over the surface of an LB agar plate: 40 μlof 20 mg/ml X-gal (or Bluo-gal) in DMF plus 4 μl 200 mg/ml IPTG. Allowliquid to adsorb into agar for 3-4 hours at 37° C. before plating cells.

B. To liquid LB agar at ˜45° C., add: X-gal (or Bluo-Gal) (20 mg/ml inDMF) to make 50 μg/ml and IPTG (200 mM in water) to make 0.5-1 mM, justprior to pouring plates. Store X-gal and Bluo-Gal in a light-shieldedcontainer.

Colony color may be enhanced by placing the plates at 5° C. for a fewhours after the overnight incubation at 37° C. Protocol B can give moreconsistent colony color than A, but A is more convenient when selectionplates are needed on short notice.

Recombination in Clonase reactions continues for many hours. Whileincubations of 45-60 minutes are usually sufficient, reactions withlarge DNAs, or in which both parental DNAs are supercoiled, or whichwill be transformed into cells of low competence, can be improved withlonger incubation times, such as 2-24 hours at 25° C.

Procedure:

1. Assemble reactions as follows (combine all components at roomtemperature, except GATEWAY™ LR Clonase™ Enzyme Mix (“Clonase LR”),before removing Clonase LR from frozen storage):

Tube 1 Tube 2 Tube 3 Tube 4 Component Neg. Pos. Neg. Test p-Gate-βGal,(Posi- 4 μl 4 μl tive control Entry Clone) 75 ng/μl pDEST1 (Positivecon- 4 μl 4 μl trol Destination Vector), 75 ng/μl Your Entry Clone 1-8μl  1-8 μl  (100-300 ng) Destination Vector for 4 μl 4 μl your nucleicacid molecule, 75 ng/μl 5 X LR Reaction Buffer 4 μl 4 μl 4 μl 4 μl TE 8μl 4 μl To 20 μl    To 16 μl    GATEWAY ™ LR — 4 μl — 4 μl Clonase ™Enzyme Mix (store at −80° C., add last) Total Volume 20 μl  20 μl  20μl  20 μl 2. Remove the GATEWAY™ LR Clonase™ Enzyme Mix from the −80° C. freezer,place immediately on ice. The Clonase takes only a few minutes to thaw.3. Add 4 μl of GATEWAY™ LR Clonase™ Enzyme Mix to reactions #2 and #4;4. Return GATEWAY™ LR Clonase™ Enzyme Mix to −80° C. freezer.5. Incubate tubes at 25° for at least 60 minutes.6. Add 2 μl Clonase Stop solution to all reactions. Incubate for 20 minat 37° C. (This step usually increases the total number of coloniesobtained by 10-20 fold.)7. Transform 2 μl into 100 μl competent E. coli. Select on platescontaining ampicillin at 100 μg/ml.

Example 7 Transformation of E. coli

To introduce cloning or Expression Vectors prepared using therecombinational cloning system of the invention, any standard E. colitransformation protocol should be satisfactory. The following steps arerecommended for best results:

-   -   1. Let the mixture of competent cells and Recombinational        Cloning System reaction product stand on ice at least 15 minutes        prior to the heat-shock step. This gives time for the        recombination proteins to dissociate from the DNA, and improves        the transformation efficiency.    -   2. Expect the reaction to be about 1%-5% efficient, i.e., 2 μl        of the reaction should contain at least 100 pg of the Expression        Clone plasmid (taking into account the amounts of each parental        plasmid in the reaction, and the subsequent dilution). If the E.        coli cells have a competence of 10⁷ CFU/μg, 100 pg of the        desired clone plasmid will give about 1000 colonies, or more, if        the entire transformation is spread on one ampicillin plate.    -   3. Always do a control pUC DNA transformation. If the number of        colonies is not what you expect, the pUC DNA transformation        gives you an indication of where the problem was.

Example 8 Preparation of attB-PCR Product

For preparation of attB-PCR products in the PCR cloning methodsdescribed in Example 9 below, PCR primers containing attB1 and attB2sequences are used. The attB1 and attB2 primer sequences are as follows:

attB1:  (SEQ ID NO: 31) 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCT-(template-specific sequence)-3′ attB2:  (SEQ ID NO: 32)5′-GGGGACCACTTTGTACAAGAAAGCTGGGT-(template- specific sequence)-3′The attB1 sequence should be added to the amino primer, and the attB2sequence to the carboxy primer. The 4 guanines at the 5′ ends of each ofthese primers enhance the efficiency of the minimal 25 bp attB sequencesas substrates for use in the cloning methods of the invention.

Standard PCR conditions may be used to prepare the PCR product. Thefollowing suggested protocol employs PLATINUM Taq DNA Polymerase HighFidelity®, available commercially from Life Technologies, Inc.(Rockville, Md.). This enzyme mix eliminates the need for hot starts,has improved fidelity over Taq, and permits synthesis of a wide range ofamplicon sizes, from 200 bp to 10 kb, or more, even on genomictemplates.

Materials needed:

-   -   PLATINUM Taq DNA Polymerase High Fidelity® (Life Technologies,        Inc.)    -   attB1- and attB2-containing primer pair (see above) specific for        your template    -   DNA template (linearized plasmid or genomic DNA)    -   10× High Fidelity PCR Buffer    -   10 mM dNTP mix    -   PEG/MgCl₂ Mix (30% PEG 8000, 30 mM MgCl₂)

Procedure:

1.) Assemble the reaction as follows:

Reaction with Reaction with Component Plasmid Target Genomic Target 10XHigh Fidelity PCR Buffer 5 μl 5 μl dNTP Mix 10 mM 1 μl 1 μl MgSO₄, 50 mM2 μl 2 μl attB1 Primer, 10 μM 2 μl 1 μl attB2 Primer, 10 μM 2 μl 1 μlTemplate DNA 1-5 ng* ≧100 ng PLATINUM Taq High Fidelity 2 μl 1 μl Waterto 50 μl to 50 μl *Use of higher amounts of plasmid template may permitfewer cycles (10-15) of PCR2.) Add 2 drops mineral oil, as appropriate.

3.) Denature for 30 sec. at 94° C.

4.) Perform 25 cycles:

94° C. for 15 sec-30 sec

55° C. for 15 sec-30 sec

68° C. for 1 min per kb of template.

5.) Following the PCR reaction, apply 1-2 μl of the reaction mixture toan agarose gel, together with size standards (e.g., 1 Kb Plus Ladder,Life Technologies, Inc.) and quantitation standards (e.g., Low MassLadder, Life Technologies, Inc.), to assess the yield and uniformity ofthe product.

Purification of the PCR product is recommended, to remove attB primerdimers which can clone efficiently into the Entry Vector. The followingprotocol is fast and will remove DNA <300 bp in size:

6.) Dilute the 50 μl PCR reaction to 200 μl with TE.7.) Add 100 μl PEG/MgCl₂ Solution. Mix and centrifuge immediately at13,000 RPM for 10 min at room temperature. Remove the supernatant(pellet is clear and hard to see).8.) Dissolve the pellet in 50 μl TE and check recovery on a gel.

If the starting PCR template is a plasmid that contains the gene forKan^(r), it is advisable to treat the completed PCR reaction with therestriction enzyme DpnI, to degrade the plasmid since unreacted residualstarting plasmid is a potential source of false-positive colonies fromthe transformation of the GATEWAY™ Cloning System reaction. Adding ˜5units of DpnI to the completed PCR reaction and incubating for 15 min at37° C. will eliminate this potential problem. Heat inactivate the DpnIat 65° C. for 15 min, prior to using the PCR product in the GATEWAY™Cloning System reaction.

Example 9 Cloning attB-PCR Products into Entry Vectors Via the BP(“Gateward”) Reaction

The addition of 5′-terminal attB sequences to PCR primers allowssynthesis of a PCR product that is an efficient substrate forrecombination with a Donor (attP) Plasmid in the presence of GATEWAY™ BPClonase™ Enzyme Mix. This reaction produces an Entry Clone of the PCRproduct (See FIG. 8).

The conditions of the Gateward Cloning reaction with an attB PCRsubstrate are similar to those of the BP Reaction (see Example 10below), except that the attB-PCR product (see Example 8) substitutes forthe Expression Clone, and the attB-PCR positive control (attB-tet^(r))substitutes for the Expression Clone Positive Control (GFP).

Materials needed:

-   -   5×BP Reaction Buffer    -   Desired attB-PCR product DNA, 50-100 ng in ≦8 μl TE.    -   Donor (attP) Plasmid (FIGS. 49-54), 75 ng/μl supercoiled DNA    -   attB-tet^(r) PCR product positive control, 25 ng/μl    -   GATEWAY™ BP Clonase™ Enzyme Mix (stored at −80° C.)    -   10× Clonase Stop Solution    -   pUC19 DNA, 10 pg/μl.    -   Chemically competent E. coli cells (competence: ≧1×10⁷ CFU/μg),        400 μl

Notes:

-   -   Preparation of attB-PCR DNA: see Example 8.    -   The Positive Control attB-tet^(r)PCR product contains a        functional copy of the tee gene of pBR322, with its own        promoter. By plating the transformation of the control BP        Reaction on kanamycin (50 μg/ml) plates (if kan^(r) Donor        Plasmids are used; see FIGS. 49-52) or an alternative selection        agent (e.g., gentamycin, if gen^(r) Donor Plasmids are used; see        FIG. 54), and then picking about 50 of these colonies onto        plates with tetracycline (20 μg/ml), the percentage of Entry        Clones containing functional tet^(r) among the colonies from the        positive control reaction can be determined (% Expression        Clones=(number of tet^(r)+kan^(r) (or gen^(r)) colonies/kan^(r)        (or gen^(r)) colonies).

Procedure:

1. Assemble reactions as follows. Combine all components except GATEWAY™BP Clonase™ Enzyme Mix, before removing GATEWAY™ BP Clonase™ Enzyme Mixfrom frozen storage.

Neg. Pos. Test Component Tube 1 Tube 2 Tube 3 attB-PCR product, 50-100ng 1-8 μl  Donor (attP) Plasmid 75 ng/μl 2 μl 2 μl 2 μl attB-PCR tet^(r)control DNA (75 4 μl ng/μl) 5 X BP Reaction Buffer 4 μl 4 μl 4 μl TE 10μl  6 μl To 16 μl    GATEWAY ™ BP Clonase ™ 4 μl 4 μl 4 μl Enzyme Mix(store at −80° C., add last) Total Volume 20 μl  20 μl  20 μl 2. Remove the GATEWAY™ BP Clonase™ Enzyme Mix from the −80° C. freezer,place immediately on ice. The Clonase takes only a few minutes to thaw.3. Add 4 μl of GATEWAY™ BP Clonase™ Enzyme Mix to the subcloningreaction, mix.4. Return GATEWAY™ BP Clonase™ Enzyme Mix to −80° C. freezer.5. Incubate tubes at 25° for at least 60 minutes.6. Add 2 μl Proteinase K (2 μg/μl) to all reactions. Incubate for 20 minat 37° C.7. Transform 2 μl into 100 μl competent E. coli, as per 3.2, above.Select on LB plates containing kanamycin, 50 μg/ml.

Results:

In initial experiments, primers for amplifying tetR and ampR from pBR322were constructed containing only the tetR- or ampR-specific targetingsequences, the targeting sequences plus attB1 (for forward primers) orattB2 (for reverse primers) sequences shown in FIG. 9, or the attB1 orattB2 sequences with a 5′ tail of four guanines. The construction ofthese primers is depicted in FIG. 65. After PCR amplification of tetRand ampR from pBR322 using these primers and cloning the PCR productsinto host cells using the recombinational cloning system of theinvention, the results shown in FIG. 66 were obtained. These resultsdemonstrated that primers containing attB sequences provided for asomewhat higher number of colonies on the tetracycline and ampicillinplates. However, inclusion of the 5′ extensions of four or five guanineson the primers in addition to the attB sequences provided significantlybetter cloning results, as shown in FIGS. 66 and 67. These resultsindicate that the optimal primers for cloning of PCR products usingrecombinational cloning will contain the recombination site sequenceswith a 5′ extension of four or five guanine bases.

To determine the optimal stoichiometry between attB-containing PCRproducts and attP-containing Donor plasmid, experiments were conductedwhere the amount of PCR product and Donor plasmid were varied during theBP Reaction. Reaction mixtures were then transformed into host cells andplated on tetracycline plates as above. Results are shown in FIG. 68.These results indicate that, for optimal recombinational cloning resultswith a PCR product in the size range of the tet gene, the amounts ofattP-containing Donor plasmids are between about 100-500 ng (mostpreferably about 200-300 ng), while the optimal concentrations ofattB-containing PCR products is about 25-100 ng (most preferably about100 ng), per 20 μl reaction.

Experiments were then conducted to examine the effect of PCR productsize on efficiency of cloning via the recombinational cloning approachof the invention. PCR products containing attB1 and attB2 sites, atsizes 256 bp, 1 kb, 1.4 kb, 3.4 kb, 4.6 kb, 6.9 kb and 10.1 kb wereprepared and cloned into Entry vectors as described above, and hostcells were transformed with the Entry vectors containing the cloned PCRproducts. For each PCR product, cloning efficiency was calculatedrelative to cloning of pUC 19 positive control plasmids as follows:

${{Cloning}\mspace{14mu} {Efficiency}} = {\frac{{CFU}\text{/}{ng}{\mspace{11mu} \;}{attB}\mspace{14mu} {PCR}\mspace{14mu} {product}}{{CFU}\text{/}{ng}\mspace{14mu} {pUC19}\mspace{14mu} {control}} \times \frac{{Size}\mspace{14mu} ({kb})\mspace{14mu} {PCR}\mspace{14mu} {product}}{{Size}\mspace{14mu} ({kb})\mspace{14mu} {pUC19}\mspace{14mu} {control}}}$

The results of these experiments are depicted in FIGS. 69A-69C (for 256bp PCR fragments), 70A-70C (for 1 kb PCR fragments), 71A-71C (for 1.4 kbPCR fragments), 72A-72C (for 3.4 kb PCR fragments), 73A-73C (for 4.6 kbPCR fragments), 74 (for 6.9 kb PCR fragments), and 75-76 (for 10.1 kbPCR fragments). The results shown in these figures are summarized inFIG. 77, for different weights and moles of input PCR DNA.

Together, these results demonstrate that attB-containing PCR productsranging in size from about 0.25 kb to about 5 kb clone relativelyefficiently in the recombinational cloning system of the invention.While PCR products larger than about 5 kb clone less efficiently(apparently due to slow resolution of cointegrates), longer incubationtimes during the recombination reaction appears to improve theefficiency of cloning of these larger PCR fragments. Alternatively, itmay also be possible to improve efficiency of cloning of large (>about 5kb) PCR fragments by using lower levels of input attP Donor plasmid andperhaps attB-containing PCR product, and/or by adjusting reactionconditions (e.g., buffer conditions) to favor more rapid resolution ofthe cointegrates.

Example 0.10 The BP Reaction

One purpose of the Gateward (“Entry”) reaction is to convert anExpression Clone into an Entry Clone. This is useful when you haveisolated an individual Expression Clone from an Expression Clone cDNAlibrary, and you wish to transfer the nucleic acid molecule of interestinto another Expression Vector, or to move a population of moleculesfrom an attB or attL library. Alternatively, you may have mutated anExpression Clone and now wish to transfer the mutated nucleic acidmolecule of interest into one or more new Expression Vectors. In bothcases, it is necessary first to convert the nucleic acid molecule ofinterest to an Entry Clone.

Materials needed:

-   -   5×BP Reaction Buffer    -   Expression Clone DNA, 100-300 ng in ≦8 μl TE.    -   Donor (attP) Vector, 75 ng/μl, supercoiled DNA    -   Positive control attB-tet-PCR DNA, 25 ng/μl    -   GATEWAY™ BP Clonase™ Enzyme Mix (stored at −80° C.)    -   Clonase Stop Solution (Proteinase K, 2 μg/μl).

Notes:

Preparation of the Expression. Clone DNA: Miniprep DNA treated withRNase works well.

-   1. As with the LR Reaction (see Example 14), the BP Reaction is    strongly influenced by the topology of the reacting DNAs. In    general, the reaction is most efficient when one of the DNAs is    linear and the other is supercoiled, compared to reactions where the    DNAs are both linear or both supercoiled. Further, linearizing the    attB Expression Clone (anywhere within the vector) will usually give    more colonies than linearizing the Donor (attP) Plasmid. If finding    a suitable cleavage site within your Expression Clone vector proves    difficult, you may linearize the Donor (attP) Plasmid between the    attP1 and attP2 sites (for example, at the NcoI site), avoiding the    ccdB gene. Maps of Donor (attP) Plasmids are given in FIGS. 49-54.

Procedure:

1. Assemble reactions as follows. Combine all components at roomtemperature, except GATEWAY™ BP Clonase™ Enzyme Mix, before removingGATEWAY™ BP Clonase™ Enzyme Mix from freezer.

Neg. Pos. Test Component Tube 1 Tube 2 Tube 3 Positive Control,attB-tet-PCR DNA, 4 μl 4 μl 25 ng/μl Desired attB Expression Clone DNA1-8 μl  (100 ng) linearized Donor (attP) Plasmid, 75 ng/μl 2 μl 2 μl 2μl 5 X BP Reaction Buffer 4 μl 4 μl 4 μl TE 10 μl  6 μl To 16 μl   GATEWAY ™ BP Clonase ™ Enzyme — 4 μl 4 μl Mix (store at −80° C., addlast) Total Volume 20 μl  20 μl  20 μl 2. Remove the GATEWAY™ BP Clonase™ Enzyme Mix from the −80° C. freezer,place immediately on ice. The mixture takes only a few minutes to thaw.3. Add 4 μl of GATEWAY™ BP Clonase™ Enzyme Mix to the subcloningreaction, mix.4. Return GATEWAY™ BP Clonase™ Enzyme Mix to −80° C. freezer.5. Incubate tubes at 25° for at least 60 minutes. If both the attB andattP DNAs are supercoiled, incubation for 2-24 hours at 25° C. isrecommended.6. Add 2 μl Clonase Stop Solution. Incubate for 10 min at 37° C.7. Transform 2 μl into 100 μl competent E. coli, as above. Select on LBplates containing 50 μg/ml kanamycin.

Example 11 Cloning PCR Products into Entry Vectors Using StandardCloning Methods Preparation of Entry Vectors for Cloning of PCR Products

All of the Entry Vectors of the invention contain the death gene ccdB asa stuffer between the “left” and “right” restriction sites. Theadvantage of this arrangement is that there is virtually no backgroundfrom vector that has not been cut with both restriction enzymes, becausethe presence of the ccdB gene will kill all standard E. coli strains.Thus it is necessary to cut each Entry Vector twice, to remove the ccdBfragment.

We strongly recommend that, after digestion of the Entry Vector with thesecond restriction enzyme, you treat the reaction with phosphatase (calfintestine alkaline phosphatase, CIAP or thermosensitive alkalinephosphatase, TSAP). The phosphatase can be added directly to thereaction mixture, incubated for an additional time, and inactivated.This step dephosphorylates both the vector and ccdB fragments, so thatduring subsequent ligation there is less competition between the ccdBfragment and the DNA of interest for the termini of the Entry Vector.

Blunt Cloning of PCR products

Generally PCR products do not have 5′ phosphates (because the primersare usually 5′ OH), and they are not necessarily blunt. (On this latterpoint, see Brownstein, et al., BioTechniques 20: 1006, 1996 for adiscussion of how the sequence of the primers affects the addition ofsingle 3′ bases.) The following protocol repairs these two defects.

In a 0.5 ml tube, ethanol precipitate about 40 ng of PCR product (asjudged from an agarose gel).

-   1. Dissolve the precipitated DNA in 10 μl comprising 1 μl 10 mM    rATP, 1 μl mixed 2 mM dNTPs (i.e., 2 mM each dATP, dCTP, dTTP, and    dGTP), 2 μl 5×T4 polynucleotide kinase buffer (350 mM Tris HCl    (pH7.6), 50 mM MgCl₂, 500 mM KCl, 5 mM 2-mercaptoethanol) 10 units    T4 polynucleotide kinase, 1 μl T4 DNA polymerase, and water to 10    μl.-   2. Incubate the tube at 37° for 10 minutes, then at 65° for 15    minutes, cool, centrifuge briefly to bring any condensate to the tip    of the tube.-   3. Add 5 μl of the PEG/MgCl₂ solution, mix and centrifuge at room    temperature for 10 minutes. Discard supernatant.-   4. Dissolve the invisible precipitate in 10 μl containing 2 μl 5×T4    DNA ligase buffer (Life Technologies, Inc.), 0.5 units T4 DNA    ligase, and about 50 ng of blunt, phosphatase-treated Entry Vector.    5. Incubate at 25° for 1 hour, then 65° for 10 minutes. Add 90 μl    TE, transform 10 μl into 50-100 μl competent E. coli cells.    6. Plate on kanamycin.

Note:

In the above protocol, steps b-c simultaneously polish the ends of thePCR product (through the exonuclease and polymerase activities of T4 DNApolymerase) and phosphorylate the 5′ ends (using T4 polynucleotidekinase). It is necessary to inactivate the kinase, so that the blunt,dephosphorylated vector in step e cannot self ligate. Step d (the PEGprecipitation) removes all small molecules (primers, nucleotides), andhas also been found to improve the yield of cloned PCR product by 50fold.

Cloning PCR Products after Digestion with Restriction Enzymes

Efficient cloning of PCR products that have been digested withrestriction enzymes includes three steps: inactivation of Taq DNApolymerase, efficient restriction enzyme cutting, and removal of smallDNA fragments.

Inactivation of Taq DNA Polymerase:

Carryover of Taq DNA polymerase and dNTPs into a RE digestionsignificantly reduces the success in cloning a PCR product (D. Fox etal., FOCUS 20(1):15, 1998), because Taq DNA polymerase can fill insticky ends and add bases to blunt ends. Either TAQQUENCH™ (obtainablefrom Life Technologies, Inc.; Rockville, Md.) or extraction with phenolcan be used to inactivate the Taq.

Efficient Restriction Enzyme Cutting:

Extra bases on the 5′ end of each PCR primer help the RE cut near endsof PCR products. With the availability of cheap primers, adding 6 to 9bases on the 5′ sides of the restriction sites is a good investment toensure that most of the ends are digested. Incubation of the DNA with a5-fold excess of restriction enzyme for an hour or more helps ensuresuccess.

Removal of Small Molecules Before Ligation:

Primers, nucleotides, primer dimers, and small fragments produced by therestriction enzyme digestion, can all inhibit or compete with thedesired ligation of the PCR product to the cloning vector. This protocoluses PEG precipitation to remove small molecules.

Protocol for Cutting the Ends of PCR Products with RestrictionEnzyme(s):

1. Inactivation of Taq DNA Polymerase in the PCR Product:

Option A: Extraction with Phenol

-   -   A1. Dilute the PCR reaction to 200 μl with TB. Add an equal        volume of phenol:chloroform:isoamyl alcohol, vortex vigorously        for 20 seconds, and centrifuge for 1 minute at room temperature.        Discard the lower phase.    -   A2. Extract the phenol from the DNA and concentrate as follows.        Add an equal volume of 2-butanol (colored red with “Oil Red O”        from Aldrich, if desired), vortex briefly, centrifuge briefly at        room temperature. Discard the upper butanol phase. Repeat the        extraction with 2-butanol. This time the volume of the lower        aqueous phase should decrease significantly. Discard the upper        2-butanol phase.    -   A3. Ethanol precipitate the DNA from the aqueous phase of the        above extractions. Dissolve in a 200 μl of a suitable        restriction enzyme (RE) buffer.        Option B: Inactivation with TaqQuench    -   B1. Ethanol precipitate an appropriate amount of PCR product        (100 ng to 1 μg), dissolve in 200 μl of a suitable RE buffer.    -   B2. Add 2 μl TaqQuench.        2. Add 10 to 50 units of restriction enzyme and incubate for at        least 1 hour. Ethanol precipitate if necessary to change buffers        for digestion at the other end of the PCR product.        3. Add ½ volume of the PEG/MgCl₂ mix to the RE digestion. Mix        well and immediately centrifuge at room temperature for 10        minutes. Discard the supernatant (pellet is usually invisible),        centrifuge again for a few seconds, discard any remaining        supernatant.        4. Dissolve the DNA in a suitable volume of TE (depending on the        amount of PCR product in the original amplification reaction)        and apply an aliquot to an agarose gel to confirm recovery.        Apply to the same gel 20-100 ng of the appropriate Entry Vector        that will be used for the cloning.

Example 12 Determining the Expected Size of the GATEWAY™ CloningReaction Products

If you have access to a software program that will electronically cutand splice sequences, you can create electronic clones to aid you inpredicting the sizes and restriction patterns of GATEWAY™ Cloning Systemrecombination products.

The cleavage and ligation steps performed by the enzyme Int in theGATEWAY™ Cloning System recombination reactions mimic a restrictionenzyme cleavage that creates a 7-bp 5′-end overhang followed by aligation step that reseals the ends of the daughter molecules. Therecombination proteins present in the Clonase cocktails (see Example 19below) recognize the 15 bp core sequence present within all four typesof att sites (in addition to other flanking sequences characteristic ofeach of the different types of att sites).

By treating these sites in your software program as if they wererestriction sites, you can cut and splice your Entry Clones with variousDestination Vectors and obtain accurate maps and sequences of theexpected results from your GATEWAY™ Cloning System reactions.

Example 13 Protein Expression Brief Review of Protein Expression

Transcription:

The most commonly used promoters in E. coli Expression Vectors arevariants of the lac promoter, and these can be turned on by adding IPTGto the growth medium. It is usually good to keep promoters off untilexpression is desired, so that the host cells are not made sick by theoverabundance of some heterologous protein. This is reasonably easy inthe case of the lac promoters used in E. coli. One needs to supply thelac I gene (or its more productive relative, the lac I^(q) gene) to makelac repressor protein, which binds near the promoter and keepstranscription levels low. Some Destination Vectors for E. coliexpression carry their own lac I^(q) gene for this purpose. (However,lac promoters are always a little “on,” even in the absence of IPTG.)

Controlling transcription in eukaryotic cells is not nearly sostraightforward or efficient. The tetracycline system of Bujard andcolleagues is the most successful approach, and one of the DestinationVectors (pDEST11; FIG. 31) has been constructed to supply this function.

Translation:

Ribosomes convert the information present in mRNA into protein.Ribosomes scan RNA molecules looking for methionine (AUG) codons, whichbegin nearly all nascent proteins. Ribosomes must, however, be able todistinguish between AUG codons that code for methionine in the middle ofproteins from those at the start. Most often ribosomes choose AUGs thatare 1) first in the RNA (toward the 5′ end), and 2) have the propersequence context. In E. coli the favored context (first recognized byShine and Dalgarno, Eur. J. Biochem. 57: 221 (1975)) is a run of purines(As and Gs) from five to 12 bases upstream of the initiating AUG,especially AGGAGG or some variant.

In eukaryotes, a survey of translated mRNAs by Kozak (J. Biol. Chem.266: 19867 (1991)) has revealed a preferred sequence context, gcc AccATGG, around the initiating methionine, with the A at −3 being mostimportant, and a purine at +4 (where the A of the ATG is +1), preferablya G, being next most influential. Having an A at −3 is enough to makemost ribosomes choose the first AUG of an mRNA, in plants, insects,yeast, and mammals. (For a review of initiation of protein synthesis ineukaryotic cells, see: Pain, V. M. Eur. J. Biochem. 236:747-771, 1996.)

Consequences of Translation Signals for GATEWAY™ Cloning System:

First, translation signals (Shine-Dalgarno in E. coli, Kozak ineukaryotes) have to be close to the initiating ATG. The attB site is 25base pairs long. Thus if translation signals are desired near thenatural ATG of the nucleic acid molecule of interest, they must bepresent in the Entry Clone of that nucleic acid molecule of interest.Also, when a nucleic acid molecule of interest is moved from an EntryClone to a Destination vector, any translation signals will move along.The result is that the presence or absence of Shine-Dalgarno and/orKozak sequences in the Entry Clone must be considered, with the eventualDestination Vectors to be used in mind.

Second, although ribosomes choose the 5′ ATG most often, internal ATGsare also used to begin protein synthesis. The better the translationcontext around this internal ATG, the more internal translationinitiation will be seen. This is important in the GATEWAY™ CloningSystem, because you can make an Entry Clone of your nucleic acidmolecule of interest, and arrange to have Shine-Dalgarno and/or Kozaksequences near the ATG. When this cassette is recombined into aDestination Vector that transcribes your nucleic acid molecule ofinterest, you get native protein. If you want, you can make a fusionprotein in a different Destination Vector, since the Shine-Dalgarnoand/or Kozak sequences do not contain any stop signals in the samereading frame. However, the presence of these internal translationsignals may result in a significant amount of native protein being made,contaminating, and lowering the yield of, your fusion protein. This isespecially likely with short fusion tags, like His6.

A good compromise can be recommended. If an Entry Vector like pENTR7(FIG. 16) or pENTR8 (FIG. 17) is chosen, the Kozak bases are present fornative eukaryotic expression. The context for E. coli translation ispoor, so the yield of an amino-terminal fusion should be good, and thefusion protein can be digested with the TEV protease to make near-nativeprotein following purification.

Recommended Conditions for Synthesis of Proteins in E. coli:

When making proteins in E. coli it is advisable, at least initially, toincubate your cultures at 30° C., instead of at 37° C. Our experienceindicates that proteins are less likely to form aggregates at 30° C. Inaddition, the yields of proteins from cells grown at 30° C. frequentlyare improved.

The yields of proteins that are difficult to express may also beimproved by inducing the cultures in mid-log phase of growth, usingcultures begun in the morning from overnight growths, as opposed toharvesting directly from an overnight culture. In the latter case, thecells are preferably in late log or stationary growth, which can favorthe formation of insoluble aggregates.

Example 14 Constructing Destination Vectors from Existing Vectors

Destination Vectors function because they have two recombination sites,attR1 and attR2, flanking a chloramphenicol resistance (CmR) gene and adeath gene, ccdB. The GATEWAY™ Cloning System recombination reactionsexchange the entire Cassette (except for a few bases comprising part ofthe attB sites) for the DNA segment of interest from the Entry Vector.Because attR1, CmR, ccdB gene, and attR2 are contiguous, they can bemoved on a single DNA segment. If this Cassette is cloned into aplasmid, the plasmid becomes a Destination Vector. FIG. 63 shows aschematic of the GATEWAY™ Cloning System Cassette; attR cassettes in allthree reading frames contained in vectors pEZC15101, pEZC15102 andpEZC15103 are shown in FIGS. 64A, 64B, and 64C, respectively.

The protocol for constructing a Destination Vector is presented below.Keep in mind the following points:

-   -   Destination Vectors must be constructed and propagated in one of        the DB strains of E. coli (e.g., DB3.1, and particularly E. coli        LIBRARY EFFICIENCY® DB3.1™ Competent Cells) available from Life        Technologies, Inc. (and described in detail in U.S. Provisional        Application No. 60/122,392, filed on Mar. 2, 1999, which is        incorporated herein by reference), because the ccdB death gene        will kill any E. coli strain that has not been mutated such that        it will survive the presence of the ccdB gene.    -   If your Destination Vector will be used to make a fusion        protein, a GATEWAY™ Cloning System cassette with the correct        reading frame must be used. The nucleotide sequences of the ends        of the cassettes are shown in FIG. 78. The reading frame of the        fusion protein domain must be in frame with the core region of        the attR1 site (for an amino terminal fusion) so that the six As        are translated into two lysine codons. For a C-terminal fusion        protein, translation through the core region of the attR2 site        should be in frame with -TAC-AAA-, to yield -Tyr-Lys-.    -   Note that each reading frame Cassette has a different unique        restriction site between the chloramphenicol resistance and ccdB        genes (MluI for reading frame A, BglII for reading frame B, and        XbaI for reading frame C; see FIG. 63).    -   Most standard vectors can be converted to Destination Vectors,        by inserting the Entry Cassette into the MCS of that vector.

Protocol for Making a Destination Vector

1. If the vector will make an amino fusion protein, it is necessary tokeep the “aaa aaa” triplets in attR1 in phase with the triplets of thefusion protein. Determine which Entry cassette to use as follows:

-   -   a.) Write out the nucleotide sequence of the existing vector        near the restriction site into which the Entry cassette will be        cloned. These must be written in triplets corresponding to the        amino acid sequence of the fusion domain.    -   b.) Draw a vertical line through the sequence that corresponds        to the restriction site end, after it has been cut and made        blunt, i.e., after filling in a protruding 5′ end or polishing a        protruding 3′ end.    -   c.) Choose the appropriate reading frame cassette:    -   If the coding sequence of the blunt end ends after a complete        codon triplet, use the reading frame A cassette. See FIGS. 78,        79 and 80.        -   If the coding sequence of the blunt end ends in a single            base, use the reading frame B cassette. See FIGS. 78, 79 and            81.        -   If the coding sequence of the blunt end ends in two bases,            use the reading frame C cassette. See FIGS. 78, 79, 82A-B,            and 83A-C.            2. Cut one to five micrograms of the existing plasmid at the            position where you wish your nucleic acid molecule of            interest (flanked by att sites) to be after the            recombination reactions. Note: it is better to remove as            many of the MCS restriction sites as possible at this step.            This makes it more likely that restriction enzyme sites            within the GATEWAY™ Cloning System Cassette will be unique            in the new plasmid, which is important for linearizing the            Destination Vector (Example 14, below).            3. Remove the 5′ phosphates with alkaline phosphatase. While            this is not mandatory, it increases the probability of            success.            4. Make the end(s) blunt with fill-in or polishing            reactions. For example, to 1 μg of restriction enzyme-cut,            ethanol-precipitated vector DNA, add:    -   i. 20 μl 5×T4 DNA Polymerase Buffer (165 mM Tris-acetate (pH        7.9), 330 mM Na acetate, 50 mM Mg acetate, 500 μg/ml BSA, 2.5 mM        DTT)

-   ii. 5 μl 10 mM dNTP mix    -   iii. 1 Unit of T4 DNA Polymerase    -   iv. Water to a final volume of 100    -   v. Incubate for 15 min at 37° C.        5. Remove dNTPs and small DNA fragments: Ethanol precipitate        (add three volumes of room temperature ethanol containing 0.1 M        sodium acetate, mix well, immediately centrifuge at room        temperature 5-10 minutes), dissolve wet precipitate in 200 μl        TE, add 100 μl 30% PEG 8000, 30 mM MgCl₂, mix well, immediately        centrifuge for 10 minutes at room temperature, discard        supernatant, centrifuge again a few seconds, discard any        residual liquid.        6. Dissolve the DNA to a final concentration of 10-50 ng per        microliter. Apply 20-100 ng to a gel next to supercoiled plasmid        and linear size standards to confirm cutting and recovery. The        cutting does not have to be 100% complete, since you will be        selecting for the chloramphenicol marker on the Entry cassette.        7. In a 10 μl ligation reaction combine 10-50 ng vector, 10-20        ng of Entry Cassette (FIG. 79), and 0.5 units T4 DNA ligase in        ligase buffer. After one hour (or overnight, whichever is most        convenient), transform 1 μl into one of the DB strains of        competent E. coli cells with a gyrA462 mutation (See U.S.        Provisional Application No. 60/122,392, filed on Mar. 2, 1999,        which is incorporated herein by reference), preferably DB3.1,        and most preferably E. coli LIBRARY EFFICIENCY® DB3.1™ Competent        Cells. The ccdB gene on the Entry Cassette will kill other        strains of E. coli that have not been mutated so as to survive        the presence of the ccdB gene.        8. After expression in SOC medium, plate 10 μl and 100 μl on        chloramphenicol-containing (30 μg/ml) plates, incubate at 37° C.        9. Pick colonies, make miniprep DNA. Treat the miniprep with        RNase A and store in TE. Cut with the appropriate restriction        enzyme to determine the orientation of the Cassette. Choose        clones with the attR1 site next to the amino end of the protein        expression function of the plasmid.

Notes on Using Destination Vectors

-   -   We have found that about ten-fold more colonies result from a        GATEWAY™ Cloning System reaction if the Destination Vector is        linear or relaxed. If the competent cells you use are highly        competent (>10⁸ per microgram), linearizing the Destination        Vector is less essential.    -   The site or sites used for the linearization must be within the        Entry Cassette. Sites that cut once or twice within each        cassette are shown in FIGS. 80-82.    -   Minipreps of Destination Vectors will work fine, so long as they        have been treated with RNase. Since most DB strains are        endA-(See U.S. Provisional Application No. 60/122,392, filed on        Mar. 2, 1999, which is incorporated herein by reference),        minipreps can be digested with restriction enzymes without a        prior phenol extraction.    -   Reading the OD₂₆₀ of miniprep DNA is inaccurate unless the RNA        and ribonucleotides have been removed, for example, by a PEG        precipitation.

Example 15 Some Options in Choosing Appropriate Entry Vectors andDestination Vectors: An Example

In some applications, it may be desirable to express a nucleic acidmolecule of interest in two forms: as an amino-terminal fusion in E.coli, and as a native protein in eukaryotic cells. This may beaccomplished in any of several ways:

Option 1:

Your choices depend on your nucleic acid molecule of interest and thefragment that contains it, as well as the available Entry Vectors. Foreukaryotic translation, you need consensus bases according to Kozak (J.Biol. Chem. 266:19867, 1991) near the initiating methionine (ATG) codon.All of the Entry Vectors offer this motif upstream of the XmnI site(blunt cutter). One option is to amplify your nucleic acid molecule ofinterest, with its ATG, by PCR, making the amino end blunt and thecarboxy end containing the natural stop codon followed by one of the“right side” restriction sites (EcoRI, NotI, XhoI, EcoRV, or XbaI of thepENTR vectors).

If you know your nucleic acid molecule of interest does not have, forexample, an XhoI site, you can make a PCR product that has thisstructure (SEQ ID NO:33):

                           Xho I 5′ATG nnn nnn --- nnn TAA ctc gag nnn nnn 3′ 3′tac nnn nnn --- nnn att gag ctc nnn nnn 5′After cutting with XhoI, the fragment is ready to clone:

5′ ATG nnn nnn --- nnn TAA c 3′ 3′ tac nnn nnn --- nnn att gag ct 5′(If you follow this example, don't forget to put a phosphate on theamino oligo.)

Option 2:

This PCR product could be cloned into two Entry Vectors to give thedesired products, between the XmnI and XhoI sites: pENTR1A (FIGS. 10A,10B) or pENTR7 (FIGS. 16A, 16B). If you clone into pENTR1A, aminofusions will have the minimal number of amino acids between the fusiondomain and your nucleic acid molecule of interest, but the fusion cannotbe removed with TEV protease. The converse is true of clones in pENTR7,i.e., an amino fusion can be cleaved with TEV protease, at the cost ofmore amino acids between the fusion and your nucleic acid molecule ofinterest.

In this example, let us choose to clone our hypothetical nucleic acidmolecule of interest into pENTR7, between the XmnI and XhoI sites. Oncethis is accomplished, several optional protocols using the Entry ClonepENTR7 may be followed:

Option 3:

Since the nucleic acid molecule of interest has been amplified with PCR,it may be desirable to sequence it. To do this, transfer the nucleicacid molecule of interest from the Entry Vector into a vector that haspriming sites for the standard sequencing primers. Such a vector ispDEST6 (FIGS. 26A, 26B). This Destination Vector places the nucleic acidmolecule of interest in the opposite orientation to the lac promoter(which is leaky—see Example 3 above). If the gene product is toxic to E.coli, this Destination Vector will minimize its toxicity.

Option 4:

While the sequencing is going on, you might wish to check the expressionof the nucleic acid molecule of interest in, for example, CHO cells, byrecombining the nucleic acid molecule of interest into a CMV promotervector (pDEST7, FIG. 27; or pDEST12, FIG. 32), or into a baculovirusvector (pDEST8, FIG. 28; or pDEST10, FIG. 30) for expression in insectcells. Both of these vectors will transcribe the coding sequence of yournucleic acid molecule of interest, and translate it from the ATG of thePCR product using the Kozak bases upstream of the XmnI site.

Option 5:

If you wish to purify protein, for example to make antibodies, you canclone the nucleic acid molecule of interest into a His6 fusion vector,pDEST2 (FIG. 22). Since the nucleic acid molecule of interest is cloneddownstream of the TEV protease cleavage domain of pENTR7 (FIG. 16), theamino acid sequence of the protein produced will be:

(SEQ ID NO: 34)               [------attB1-----] TEV proteaseNH2-MSYYHHHHHHGITSLYKKAGF

↓

TM----COOH

The attB site and the restriction sites used to make the Destination andEntry Vectors are translated into the underlined 11 amino acids(GITSLYKKAGF) (SEQ ID NO:35). Cleavage with TEV protease (arrow) leavestwo amino acids, GT, on the amino end of the gene product.

See FIG. 55 for an example of a nucleic acid molecule of interest, thechloramphenicol acetyl transferase (CAT) gene, cloned into pENTR7 (FIG.16) as a blunt (amino)-XhoI (carboxy) fragment, then cloned byrecombination into the His6 fusion vector pDEST2 (FIG. 22).

Option 6:

If the His6 fusion protein is insoluble, you may go on and try a GSTfusion. The appropriate Destination vector is pDEST3 (FIG. 23).

Option 7:

If you need to make RNA probes and prefer SP6 RNA polymerase, you canmake the top strand RNA with your nucleic acid molecule of interestcloned into pSPORT+ (pDEST5 (FIGS. 25A, 25B)), and the bottom strand RNAwith the nucleic acid molecule of interest cloned into pSPORT(−) (pDEST6(FIGS. 26A, 26B)). Opposing promoters for Ti RNA polymerase and SP6 RNApolymerase are also present in these clones.

Option 8:

It is often worthwhile to clone your nucleic acid molecule of interestinto a variety of Destination Vectors in the same experiment. Forexample, if the number of colonies varies widely when the variousrecombination reactions are transformed into E. coli, this may be anindication that the nucleic acid molecule of interest is toxic in somecontexts. (This problem is more clearly evident when a positive controlgene is used for each Destination Vector.) Specifically, if many morecolonies are obtained when the nucleic acid molecule of interest isrecombined into pDEST6 than in pDEST5, there is a good chance thatleakiness of the lac promoter is causing some expression of the nucleicacid molecule of interest in pSPORT “+” (which is not harmful in pDEST6because the nucleic acid molecule of interest is in the oppositeorientation).

Example 16 Demonstration of a One-Tube Transfer of a PCR Product (orExpression Clone) to Expression Clone Via a Recombinational CloningReaction

In the BxP recombination (Entry or Gateward) reaction described herein,a DNA segment flanked by attB1 and attB2 sites in a plasmid conferringampicillin resistance was transferred by recombination into an attPplasmid conferring kanamycin resistance, which resulted in a productmolecule wherein the DNA segment was flanked by attL sites (attL1 andattL2). This product plasmid comprises an “attL Entry Clone” molecule,because it can react with a “attR Destination Vector” molecule via theLxR (Destination) reaction, resulting in the transfer of the DNA segmentto a new (ampicillin resistant) vector. In the previously describedexamples, it was necessary to transform the BxP reaction products intoE. coli, select kanamycin resistant colonies, grow those colonies inliquid culture, and prepare miniprep DNA, before reacting this DNA witha Destination Vector in an LxR reaction.

The goal of the following experiment was to eliminate the transformationand miniprep DNA steps, by adding the BxP Reaction products directly toan LxR Reaction. This is especially appropriate when the DNA segmentflanked by attB sites is a PCR product instead of a plasmid, because thePCR product cannot give ampicillin-resistant colonies upontransformation, whereas attB plasmids (in general) carry an ampicillinresistance gene. Thus use of a PCR product flanked by attB sites in aBxP Reaction allows one to select for the ampicillin resistance encodedby the desired attB product of a subsequent LxR Reaction.

Two reactions were prepared: Reaction A, negative control, no attB PCRproduct, (8 μl) contained 50 ng pEZC7102 (attP Donor plasmid, conferskanamycin resistance) and 2 μl BxP Clonase (22 ng/μl Int protein and 8ng/μl IHF protein) in BxP buffer (25 mM Tris HCl, pH 7.8, 70 mM KCl, 5mM spermidine, 0.5 mM EDTA, 250 μg/ml BSA). Reaction B (24 μl) contained150 ng pEZC7102, 6 μl BxP Clonase, and 120 ng of the attB-tet-PCRproduct in the same buffer as reaction A. The attB-tet-PCR productcomprised the tetracycline resistance gene of plasmid pBR322, amplifiedwith two primers containing either attB1 or attB2 sites, and having 4 Gsat their 5′ ends, as described earlier.

The two reactions were incubated at 25° C. for 30 minutes. Then aliquotsof these reactions were added to new components that comprised LxRReactions or appropriate controls for the LxR Reaction. Five newreactions were thus produced:

Reaction 1:

5 μl of reaction A was added to a 5 μl LxR Reaction containing 25 ngNcoI-cut pEZC8402 (the attR Destination Vector plasmid) in LxR buffer(37.5 mM Tris HCl, pH 7.7, 16.5 mM NaCl, 35 mM KCl, 5 mM spermidine, 375μg/ml BSA), and 1 μl of GATEWAY™ LR Clonase™ Enzyme Mix (total volume of10 μl).

Reaction 2:

Same as reaction 1, except 5 μl of reaction B (positive) were addedinstead of reaction A (negative).

Reaction 3:

Same as reaction 2, except that the amounts of Nco-cut pEZC8402 andGATEWAY™ LR Clonase™ Enzyme Mix were doubled, to 50 ng and 2 μl,respectively.

Reaction 4:

Same as reaction 2, except that 25 ng of pEZ11104 (a positive controlattL Entry Clone plasmid) were added in addition to the aliquot ofreaction B.

Reaction 5:

Positive control LxR Reaction, containing 25 ng NcoI-cut pEZC8402, 25 ngpEZ11104, 37.5 mM Tris HCl pH 7.7, 16.5 mM NaCl, 35 mM KCl, 5 mMspermidine, 375 μg/ml BSA and 1 μl GATEWAY™ LR Clonase™ Enzyme Mix in atotal volume of 5 μl.

All five reactions were incubated at 25° C. for 30 minutes. Then, 1 μlaliquots of each of the above five reactions, plus 1 μl from theremaining volume of Reaction B, the standard BxP Reaction, were used totransform 50 μl competent DH5α E. coli. DNA and cells were incubated onice for 15 min., heat shocked at 42° C. for 45 sec., and 450 μl SOC wereadded. Each tube was incubated with shaking at 37° C. for 60 min.Aliquots of 100 μl and 400 μl of each transformation were plated on LBplates containing either 50 μg/ml kanamycin or 100 μg/ml ampicillin (seeTable 2). A transformation with 10 pg of pUC19 DNA (plated on LB-amp₁₀₀)served as a control on the transformation efficiency of the DH5α cells.Following incubation overnight at 37° C., the number of colonies on eachplate was determined.

Results of these reactions are shown in Table 2.

TABLE 2 Reaction No: 1 2 3 4 5 6 Number of Colonies Vol. Neg. 1X 2X LxRLxR BxP plated: Control pEZC8402 pEZC8402 Reaction Reaction Reaction BxPand LR and LR with Pos. alone alone Reaction Clonase^( ™) Clonase^( ™)Control DNA 100 μl 2  1  8  9 ~1000 ~1000 400 μl 5 10 35 62 >2000 >2000Selection: Kan Amp Amp Amp Amp Kan *(Transformation with pUC 19 DNAyielded 1.4 × 10⁹ CFU/μg DNA.)

34 of the 43 colonies obtained from Reaction 3 were picked into 2 mlTerrific Broth with 100 μg/ml ampicillin and these cultures were grownovernight, with shaking, at 37° C. 27 of the 34 cultures gave at leastmoderate growth, and of these 24 were used to prepare miniprep DNA,using the standard protocol. These 24 DNAs were initially analyzed assupercoiled (SC) DNA on a 1% agarose gel to identify those with insertsand to estimate the sizes of the inserts. Fifteen of the 24 samplesdisplayed SC DNA of the size predicted (5553 bp) if tetx7102 hadcorrectly recombined with pEZC8402 to yield tetx8402. One of thesesamples contained two plasmids, one of ˜5500 bp and a one of ˜3500 bp.The majority of the remaining clones were approximately 4100 bp in size.

All 15 of the clones displaying SC DNA of predicted size (˜5500 bp) wereanalyzed by two different double digests with restriction endonucleasesto confirm the structure of the expected product: tetx8402. (See plasmidmaps, FIGS. 57-59) In one set of digests, the DNAs were treated with NotI and Eco RI, which should cut the predicted product just outside bothattB sites, releasing the tee insert on a fragment of 1475 bp. In thesecond set of digests, the DNAs were digested with NotI and with NruI.NruI cleaves asymmetrically within the subcloned tee insert, andtogether with NotI will release a fragment of 1019 bp.

Of the 15 clones analyzed by double restriction digestion, 14 revealedthe predicted sizes of fragments for the expected product.

Interpretation:

The DNA components of Reaction B, pEZC7102 and attB-tet-PCR, are shownin FIG. 56. The desired product of BxP Reaction B is tetx7102, depictedin FIG. 57. The LxR Reaction recombines the product of the BxP Reaction,tetx7102 (FIG. 57), with the Destination Vector, pEZC8402, shown in FIG.58. The LxR Reaction with tetx7102 plus pEZC8402 is predicted to yieldthe desired product tetx8402, shown in FIG. 59.

Reaction 2, which combined the BxP Reaction and LxR Reaction, gave fewcolonies beyond those of the negative control Reaction. In contrast,Reaction 3, with twice the amount of pEZC8402 (FIG. 58) and LxR Clonase,yielded a larger number of colonies. These colonies were analyzedfurther, by restriction digestion, to confirm the presence of expectedproduct. Reaction 4 included a known amount of attL Entry Clone plasmidin the combined BxP-plus-LxR reaction. But reaction 4 yielded only about1% of the colonies obtained when the same DNA was used in a LxR reactionalone, Reaction 6. This result suggests that the LxR reaction may beinhibited by components of the BxP reaction.

Restriction endonuclease analysis of the products of Reaction 3 revealedthat a sizeable proportion of the colonies (14 of the 34 analyzed)contained the desired tee subclone, tetx8402 (FIG. 59).

The above results establish the feasibility of performing first a BxPrecombination reaction followed by a LxR recombination reaction—in thesame tube—simply by adding the appropriate buffer mix, recombinationproteins, and DNAs to a completed BxP reaction. This method should proveuseful as a faster method to convert attB-containing PCR products intodifferent Expression Clones, eliminating the need to isolate first theintermediate attL-PCR insert subclones, before recombining these withDestination Vectors. This may prove especially valuable for automatedapplications of these reactions.

This same one-tube approach allows for the rapid transfer of nucleicacid molecules contained in attB plasmid clones into new functionalvectors as well. As in the above examples, attL subclones generated in aBxP Reaction can be recombined directly with various Destination Vectorsin a LxR reaction. The only additional requirement for using attBplasmids, instead of attB-containing PCR products, is that theDestination Vector(s) employed must contain a different selection markerfrom the one present on the attB plasmid itself and the attP vector.

Two alternative protocols for a one-tube reaction have also provenuseful and somewhat more optimal than the conditions described above.

Alternative 1:

Reaction buffer contained 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.25 mMEDTA, 2.5 mM spermidine, and 200 μg/ml BSA. After a 16 (or 3) hourincubation of the PCR product (100 ng)+attP Donor plasmid (100ng)+GATEWAY™ BP Clonase™ Enzyme Mix+Destination Vector (100 ng), 2 μl ofGATEWAY™ LR Clonase™ Enzyme Mix (per 10 μl reaction mix) was added andthe mixture was incubated an additional 6 (or 2) hours at 25° C. Stopsolution was then added as above and the mixture was incubated at 37° C.as above and transformed by electroporation with 1 μl directly intoelectrocompetent host cells. Results of this series of experimentsdemonstrated that longer incubation times (16 hours vs. 3 hours for theBP Reaction, 6 hours vs. 2 hours for the LR Reaction) resulted in abouttwice as many colonies being obtained as for the shorter incubationtimes. With two independent genes, 10/10 colonies having the correctcloning patterns were obtained.

Alternative 2:

A standard BP Reaction under the reaction conditions described above forAlternative 1 was performed for 2 hours at 25° C. Following the BPReaction, the following components were added to the reaction mixture ina total volume of 7 μl:

-   -   20 mM Tris-HCl, pH 7.5    -   100 mM NaCl    -   5 μg/ml Xis-His6    -   15% glycerol    -   ˜1000 ng of Destination Vector        The reaction mixture was then incubated for 2 hours at 25° C.,        and 2.5 μl of stop solution (containing 2 μg/ml proteinase K)        was added and the mixture was incubated at 37° C. for an        additional 10 minutes. Chemically competent host cells were then        transformed with 2 μl of the reaction mixture, or        electrocompetent host cells (e.g., EMax DH10B cells; Life        Technologies, Inc.) were electroporated with 2 μl of the        reaction mixture per 25-40 μl of cells. Following        transformation, mixtures were diluted with SOC, incubated at 37°        C., and plated as described above on media selecting for the        selection markers on the Destination Vector and the Entry clone        (B×P reaction product). Analogous results to those described for        Alternative 1 were obtained with these reaction conditions—a        higher level of colonies containing correctly recombined        reaction products were observed.

Example 17 Demonstration of a One-Tube Transfer of a PCR Product (orExpression Clone) to Expression Clone Via a Recombinational CloningReaction

Single-tube transfer of PCR product DNA or Expression Clones intoExpression Clones by recombinational cloning has also been accomplishedusing a procedure modified from that described in Example 16. Thisprocedure is as follows:

-   -   Perform a standard BP (Gateward) Reaction (see Examples 9        and 10) in 20 μl volume at 25° C. for 1 hour.    -   After the incubation is over, take a 10 μl aliquot from the 20        μl total volume and add 1 μl of Proteinase K (2 mg/ml) and        incubate at 37° C. for 10 minutes. This first aliquot can be        used for transformation and gel assay of BP reaction analysis.        Plate BP reaction transformation on LB plates with Kanamycin (50        ug/ml).    -   Add the following reagents to the remaining 10 μl aliquot of the        BP reaction:        -   1 μl of 0.75 M NaCl        -   2 μl of destination vector (150 ng/μl)        -   4 μl of LR Clonase™ (after thawing and brief mixing)    -   Mix all reagents well and incubate at 25° C. for 3 hours. Stop        the reaction at the end of incubation with 1.7 μl of Proteinase        K (2 mg/ml) and incubate at 37° C. for 10 minutes.    -   Transform 2 μl of the completed reaction into 100 μl of        competent cells. Plate 100 μl and 400 μl on LB plates with        Ampicilin (100 μg/ml).

Notes:

If your competent cells are less than 10⁸ CFU/μg, and you are concernedabout getting enough colonies, you can improve the yield several fold byincubating the BP reaction for 6-20 hours. Electroporation also canyield better colony output than chemical transformation.

PCR products greater than about 5-6 kb show significantly lower cloningefficiency in the BP reaction. In this case, we recommend using longerincubation times for both BP and LR steps.

If you want to move your insert gene into several destination vectorssimultaneously, then scale up the initial BP reaction volume so that youhave a 10 μl aliquot for adding each destination vector.

Example 18 Optimization of GATEWAY™ Clonase™ Enzyme Compositions

The enzyme compositions containing Int and IHF (for BP Reactions) wereoptimized using a standard functional recombinational cloning reaction(a BP reaction) between attB-containing plasmids and attP-containingplasmids, according to the following protocol:

Materials and Methods:

Substrates:

-   -   AttP—supercoiled pDONR201    -   AttB—linear ˜1 Kb [³H]PCR product amplified from pEZC7501

Proteins:

-   -   IntH6—His₆-carboxy-tagged λ Integrase    -   IHF—Integration Host Factor

Clonase:

-   -   50 ng/μl IntH6 and 20 ng/μl IHF, admixed in 25 mM Tris-HCl (pH        7.5),    -   22 mM NaCl, 5 mM EDTA, 1 mg/ml BSA, 5 mM Spermidine, and 50%        glycerol.

Reaction Mixture (Total Volume of 40 μl):

-   -   1000 ng AttP plasmid    -   600 ng AttB [³H] PCR product    -   8 μl Clonase (400 ng IntH6, 160 ng IHF) in 25 mM Tris-HCl (pH        7.5),    -   22 mM NaCl, 5 mM EDTA, 1 mg/ml BSA, 5 mM Spermidine, 5 mM DTT.

Reaction mixture was incubated for 1 hour at 25° C., 4 μl of 2 μg/μlproteinase K was added and mixture was incubated for an additional 20minutes at 37° C. Mixture was then extracted with an equal volume ofPhenol/Chloroform/Isoamyl alcohol. The aqueous layer was then collected,and 0.1 volumes of 3 M sodium acetate and 2 volumes of cold 100% ethanolwere added. Tubes were then spun in a microcentrifuge at maximum RPM for10 minutes at room temperature. Ethanol was decanted, and pellets wererinsed with 70% ethanol and re-centrifuged as above. Ethanol wasdecanted, and pellets were allowed to air dry for 5-10 minutes and thendissolved in 20 μl of 33 mM Tris-Acetate (pH 7.8), 66 mM potassiumacetate, 10 mM magnesium acetate, 1 mM DTT, and 1 mM ATP. 2 units ofexonuclease V (e.g., Plasmid Safe; EpiCentre, Inc., Madison, Wis.) wasthen added, and the mixture was incubated at 37° C. for 30 minutes.

Samples were then TCA-washed by spotting 30 μl of reaction mixture ontoa Whatman GF/C filter, washing filters once with 10% TCA+1% NaPPi for 10minutes, three times with 5% TCA for 5 minutes each, and twice withethanol for 5 minutes each. Filters were then dried under a heat lamp,placed into a scintillation vial, and counted on a β liquidscintillation counter (LSC).

The principle behind this assay is that, after exonuclease V digestion,only double-stranded circular DNA survives in an acid-insoluble form.All DNA substrates and products that have free ends are digested to anacid-soluble form and are not retained on the filters. Therefore, onlythe ³H-labeled attB linear DNA which ends up in circular form after bothinter- and intramolecular integration is complete is resistant todigestion and is recovered as acid-insoluble product. Optimal enzyme andbuffer formulations in the Clonase compositions therefore are those thatgive the highest levels of circularized ³H-labeled attB-containingsequences, as determined by highest cpm in the LSC. Although this assaywas designed for optimization of GATEWAY™ BP Clonase™ Enzyme Mixcompositions (Int+IHF), the same type of assay may be performed tooptimize GATEWAY™ LR Clonase™ Enzyme Mix compositions (Int+IHF+Xis),except that the reaction mixtures would comprise 1000 ng of AttR(instead of AttP) and 600 ng of AttL (instead of AttB), and 40 ng ofHis₆-carboxy-tagged Xis (XisH6) in addition to the IntH6 and IHF.

Example 19 Testing Functionality of Entry and Destination Vectors

As part of assessment of the functionality of particular vectors of theinvention, it is important to functionally test the ability of thevectors to recombine. This assessment can be carried out by performing arecombinational cloning reaction (as schematized in FIGS. 2, 4, and 5Aand 5B, and as described herein and in commonly owned U.S. applicationSer. No. 08/486,139, filed Jun. 7, 1995, Ser. No. 08/663,002, filed Jun.7, 1996 (now U.S. Pat. No. 5,888,732), Ser. No. 09/005,476, filed Jan.12, 1998, and Ser. No. 09/177,387, filed Oct. 23, 1998, the disclosuresof all of which are incorporated by reference herein in theirentireties), by transforming E. coli and scoring colony forming units.However, an alternative assay may also be performed to allow faster,more simple assessment of the functionality of a given Entry orDestination Vector by agarose gel electrophoresis. The following is adescription of such an in vitro assay.

Materials and Methods:

Plasmid templates pEZC1301 (FIG. 84) and pEZC1313 (FIG. 85), eachcontaining a single wild type att site, were used for the generation ofPCR products containing attL or attR sites, respectively. Plasmidtemplates were linearized with AlwNI, phenol extracted, ethanolprecipitated and dissolved in TE to a concentration of 1 ng/μl.

PCR primers (capital letters represent base changes from wildtype):attL1 (SEQ ID NO: 36) gggg agcct gcttttttGtacAaa gttggcatta taaaaaagcattgc; attL2 (SEQ ID NO: 37)gggg agcct gctttCttGtacAaa gttggcatta taaaaaagca ttgc; attL right(SEQ ID NO: 38) tgttgccggg aagctagagt aa; attR1 (SEQ ID NO: 39)gggg Acaag ttTgtaCaaaaaagc tgaacgaga aacgtaaaat; attR2 (SEQ ID NO: 40)gggg Acaag ttTgtaCaaGaaagc tgaacgaga aacgtaaaat; attR right(SEQ ID NO: 41) ca gacggcatga tgaacctgaaPCR primers were dissolved in TE to a concentration of 500 pmol/μl.Primer mixes were prepared, consisting of attL1+attLright primers,attL2+attLright primers, attR1+attRright primers, and attR2+attRrightprimers, each mix containing 20 pmol/μl of each primer.

PCR Reactions:

1 μl plasmid template (1 ng)1 μl primer pairs (20 pmoles of each)

3 μl of H₂0 45 μl of Platinum PCR SuperMix® (Life Technologies, Inc.)

Cycling Conditions (Performed in MJ Thermocycler):

95° C./2 minutes

94° C./30 seconds

25 cycles of 58° C./30 seconds and 72° C./1.5 minutes

72° C./5 minutes

5° C./hold

The resulting attL PCR product was 1.5 kb, and the resulting attR PCRproduct was 1.0 kb.

PCR reactions were PEG/MgCl₂ precipitated by adding 150 μl H₂O and 100μl of 3×PEG/MgCl₂ solution followed by centrifugation. The PCR productswere dissolved in 50 μl of TE. Quantification of the PCR product wasperformed by gel electrophoresis of 1 μl and was estimated to be 50-100ng/μl.

Recombination reactions of PCR products containing attL or attR siteswith GATEWAY™ plasmids was performed as follows:

8 μl of H₂O

2 μl of attL or attR PCR product (100-200 ng)

2 μl of GATEWAY™ plasmid (100 ng)

4 μl of 5× Destination buffer

4 μl of GATEWAY™ LR Clonase™ Enzyme Mix

20 μl total volume (the reactions can be scaled down to a 5 μl totalvolume by adjusting the volumes of the components to about ¼ of thoseshown above, while keeping the stoichiometries the same).

Clonase reactions were incubated at 25° C. for 2 hours. 2 μl ofproteinase K (2 mg/ml) was added to stop the reaction. 10 μl was thenrun on a 1% agarose gel. Positive control reactions were performed byreacting attL1 PCR product (1.0 kb) with attR1 PCR product (1.5 kb) andby similarly reacting attL2 PCR product with attR2 PCR product toobserve the formation of a larger (2.5 kb) recombination product.Negative controls were similarly performed by reacting attL1 PCR productwith attR2 PCR product and vice versa or reactions of attL PCR productwith an attL plasmid, etc.

In alternative assays, to test attB Entry vectors, plasmids containingsingle attP sites were used. Plasmids containing single att sites couldalso be used as recombination substrates in general to test all Entryand Destination vectors (i.e., those containing attL, attR, attB andattP sites). This would eliminate the need to do PCR reactions.

Results:

Destination and Entry plasmids when reacted with appropriateatt-containing PCR products formed linear recombinant molecules thatcould be easily visualized on an agarose gel when compared to controlreactions containing no attL or attR PCR product. Thus, thefunctionality of Destination and Entry vectors constructed according tothe invention may be determined either by carrying out the Destinationor Entry recombination reactions as depicted in FIGS. 2, 4, and 5A and5B, or more rapidly by carrying out the linearization assay described inthis Example.

Example 20 PCR Cloning Using Universal Adapter-Primers

As described herein, the cloning of PCR products using the GATEWAY™ PCRCloning System (Life Technologies, Inc.; Rockville, Md.) requires theaddition of attB sites (attB1 and attB2) to the ends of gene-specificprimers used in the PCR reaction. The protocols described in thepreceding Examples suggest that the user add 29 bp (25 bp containing theattB site plus four G residues) to the gene-specific primer. It would beadvantageous to high volume users of the GATEWAY™ PCR Cloning System togenerate attB-containing PCR product using universal attBadapter-primers in combination with shorter gene-specific primerscontaining a specified overlap to the adapters. The followingexperiments demonstrate the utility of this strategy using universalattB adapter-primers and gene-specific primers containing overlaps ofvarious lengths from 6 bp to 18 bp. The results demonstrate thatgene-specific primers with overlaps of 10 bp to 18 bp can be usedsuccessfully in PCR amplifications with universal attB adapter-primersto generate full-length PCR products. These PCR products can then besuccessfully cloned with high fidelity in a specified orientation usingthe GATEWAY™ PCR Cloning System.

Methods and Results:

To demonstrate that universal attB adapter-primers can be used withgene-specific primers containing partial attB sites in PCR reactions togenerate full-length PCR product, a small 256 bp region of the humanhemoglobin cDNA was chosen as a target so that intermediate sizedproducts could be distinguished from full-length products by agarose gelelectrophoresis.

The following oligonucleotides were used:

(SEQ ID NO: 42) B1-Hgb:  GGGG ACA AGT TTG TAC AAA AAA GCA GGC T-5′-Hgb*;B2-Hgb: (SEQ ID NO: 43) GGGG ACC ACT TTG TAC AAG AAA GCT GGG T-3′-Hgb**;18B1-Hgb:  (SEQ ID NO: 44) TG TAC AAA AAA GCA GGC T-5′-Hgb; 18B2-Hgb: (SEQ ID NO: 45) TG TAC AAG AAA GCT GGG T-3′-Hgb; 15B1-Hgb:(SEQ ID NO: 46) AC AAA AAA GCA GGC T-5′-Hgb; 15B2-Hgb: (SEQ ID NO: 47)AC AAG AAA GCT GGG T-3′-Hgb; 12B1-Hgb: (SEQ ID NO: 48)AA AAA GCA GGC T-5′-Hgb; 12B2-Hgb: (SEQ ID NO: 49)AG AAA GCT GGG T-3′-Hgb; 11B1-Hgb: (SEQ ID NO: 50)A AAA GCA GGC T-5′-Hgb; 11B2-Hgb: (SEQ ID NO: 51)G AAA GCT GGG T-3′-Hgb; 10B1-Hgb: (SEQ ID NO: 52) AAA GCA GGC T-5′-Hgb;10B2-Hgb: (SEQ ID NO: 53) AAA GCT GGG T-3′-Hgb; 9B1-Hgb:AA GCA GGC T-5′-Hgb 9B2-Hgb: AA GCT GGG T-3′-Hgb 8B1-Hgb:A GCA GGC T-5′-Hgb 8B2-Hgb: A GCT GGG T-3′-Hgb 7B1-Hgb: GCA GGC T-5′-Hgb7B2-Hgb: GCT GGG T-3′-Hgb 6B1-Hgb: CA GGC T-5′-Hgb 6B2-Hgb:CT GGG T-3′-Hgb attB1 adapter: (SEQ ID NO: 54)GGGG ACA AGT TTG TAC AAA AAA GCA GGC T; attB2 adapter: (SEQ ID NO: 55)GGGG ACC ACT TTG TAC AAG AAA GCT GGG T; (SEQ ID NO: 56) *-5′-Hgb =GTC ACT AGC CTG TGG AGC AAG A; (SEQ ID NO: 57) **-3′-Hgb =AGG ATG GCA GAG GGA GAC GAC A

The aim of these experiments was to develop a simple and efficientuniversal adapter PCR method to generate attB containing PCR productssuitable for use in the GATEWAY™ PCR Cloning System. The reactionmixtures and thermocycling conditions should be simple and efficient sothat the universal adapter PCR method could be routinely applicable toany PCR product cloning application.

PCR reaction conditions were initially found that could successfullyamplify predominately full-length PCR product using gene-specificprimers containing 18 bp and 15 bp overlap with universal attB primers.These conditions are outlined below:

10 pmoles of gene-specific primers

10 pmoles of universal attB adapter-primers

1 ng of plasmid containing the human hemoglobin cDNA.

100 ng of human leukocyte cDNA library DNA.

5 μl of 10× PLATINUM Taq HiFi® reaction buffer (Life Technologies, Inc.)

2 μl of 50 mM MgSO₄

1 μl of 10 mM dNTPs

0.2 μl of PLATINUM Taq HiFi® (1.0 unit)

H₂O to 50 μl total reaction volume

Cycling Conditions:

$\left. {25 \times} \middle| \begin{matrix}{95{^\circ}\mspace{14mu} {C.\text{/}}5\mspace{14mu} \min} \\{94{^\circ}\mspace{14mu} {C.\text{/}}15\mspace{14mu} \sec} \\{50{^\circ}\mspace{14mu} {C.\text{/}}30\mspace{14mu} \sec} \\{68{^\circ}\mspace{14mu} {C.\text{/}}\text{1}\mspace{14mu} \min} \\{68{^\circ}\mspace{14mu} {C.\text{/}}5\mspace{14mu} \min} \\{5{^\circ}\mspace{14mu} {C.\text{/}}{hold}}\end{matrix} \right.$

To assess the efficiency of the method, 2 μl ( 1/25) of the 50 μl PCRreaction was electrophoresed in a 3% Agarose-1000 gel. With overlaps of12 bp or less, smaller intermediate products containing one or nouniversal attB adapter predominated the reactions. Further optimizationof PCR reaction conditions was obtained by titrating the amounts ofgene-specific primers and universal attB adapter-primers. The PCRreactions were set up as outlined above except that the amounts ofprimers added were:

0, 1, 3 or 10 pmoles of gene-specific primers0, 10, 30 or 100 pmoles of adapter-primers

Cycling Conditions:

$\left. {25 \times} \middle| \begin{matrix}{95{^\circ}\mspace{14mu} {C.\text{/}}3\mspace{14mu} \min} \\{94{^\circ}\mspace{14mu} {C.\text{/}}15\mspace{14mu} \sec} \\{50{^\circ}\mspace{14mu} {C.\text{/}}45\mspace{14mu} \sec} \\{68{^\circ}\mspace{14mu} {C.\text{/}}\text{1}\mspace{14mu} \min} \\{68{^\circ}\mspace{14mu} {C.\text{/}}5\mspace{14mu} \min} \\{5{^\circ}\mspace{14mu} {C.\text{/}}{hold}}\end{matrix} \right.$

The use of limiting amounts of gene-specific primers (3 pmoles) andexcess adapter-primers (30 pmoles) reduced the amounts of smallerintermediate products. Using these reaction conditions the overlapnecessary to obtain predominately full-length PCR product was reduced to12 bp. The amounts of gene-specific and adapter-primers was furtheroptimized in the following PCR reactions:

0, 1, 2 or 3 pmoles of gene-specific primers0, 30, 40 or 50 pmoles of adapter-primers

Cycling Conditions:

$\left. {25 \times} \middle| \begin{matrix}{95{^\circ}\mspace{14mu} {C.\text{/}}3\mspace{14mu} \min} \\{94{^\circ}\mspace{14mu} {C.\text{/}}15\mspace{14mu} \sec} \\{48{^\circ}\mspace{14mu} {C.\text{/}}1\mspace{14mu} \min} \\{68{^\circ}\mspace{14mu} {C.\text{/}}\text{1}\mspace{14mu} \min} \\{68{^\circ}\mspace{14mu} {C.\text{/}}5\mspace{14mu} \min} \\{5{^\circ}\mspace{14mu} {C.\text{/}}{hold}}\end{matrix} \right.$

The use of 2 pmoles of gene-specific primers and 40 pmoles ofadapter-primers further reduced the amounts of intermediate products andgenerated predominately full-length PCR products with gene-specificprimers containing an 11 bp overlap. The success of the PCR reactionscan be assessed in any PCR application by performing a no adaptercontrol. The use of limiting amounts of gene-specific primers shouldgive faint or barely visible bands when 1/25 to 1/10 of the PCR reactionis electrophoresed on a standard agarose gel. Addition of the universalattB adapter-primers should generate a robust PCR reaction with a muchhigher overall yield of product.

PCR products from reactions using the 18 bp, 15 bp, 12 bp, 11 bp and 10bp overlap gene-specific primers were purified using the CONCERT® RapidPCR Purification System (PCR products greater than 500 bp can be PEGprecipitated). The purified PCR products were subsequently cloned intoan attP containing plasmid vector using the GATEWAY™ PCR Cloning System(Life Technologies, Inc.; Rockville, Md.) and transformed into E. coli.Colonies were selected and counted on the appropriate antibiotic mediaand screened by PCR for correct inserts and orientation.

Raw PCR products (unpurified) from the attB adapter PCR of a plasmidclone of part of the human beta-globin (Hgb) gene were also used inGATEWAY™ PCR Cloning System reactions. PCR products generated with thefull attB B1/B2-Hgb, the 12B1/B2, 11B1/B2 and 10B1/B2 attB overlap Hgbprimers were successfully cloned into the GATEWAY™ pENTR21 attP vector(FIG. 49). 24 colonies from each (24×4=96 total) were tested and eachwas verified by PCR to contain correct inserts. The cloning efficiencyexpressed as cfu/ml is shown below:

Primer Used cfu/ml Hgb full attB 8,700 Hgb 12 bp overlap 21,000 Hgb 11bp overlap 20,500 Hgb 10 bp overlap 13,500 GFP control 1,300

Interestingly, the overlap PCR products cloned with higher efficiencythan did the full attB PCR product. Presumably, and as verified byvisualization on agarose gel, the adapter PCR products were slightlycleaner than was the full attB PCR product. The differences in colonyoutput may also reflect the proportion of PCR product molecules withintact attB sites.

Using the attB adapter PCR method, PCR primers with 12 bp attB overlapswere used to amplify cDNAs of different sizes (ranging from 1 to 4 kb)from a leukocyte cDNA library and from first strand cDNA prepared fromHeLa total RNA. While three of the four cDNAs were able to be amplifiedby this method, a non-specific amplification product was also observedthat under some conditions would interfere with the gene-specificamplification. This non-specific product was amplified in reactionscontaining the attB adapter-primers alone without any gene-specificoverlap primers present. The non-specific amplification product wasreduced by increasing the stringency of the PCR reaction and loweringthe attB adapter PCR primer concentration.

These results indicate that the adapter-primer PCR approach described inthis Example will work well for cloned genes. These results alsodemonstrate the development of a simple and efficient method to amplifyPCR products that are compatible with the GATEWAY™ PCR Cloning Systemthat allows the use of shorter gene-specific primers that partiallyoverlap universal attB adapter-primers. In routine PCR cloningapplications, the use of 12 bp overlaps is recommended. The methodsdescribed in this Example can thus reduce the length of gene-specificprimers by up to 17 residues or more, resulting in a significant savingsin oligonucleotide costs for high volume users of the GATEWAY™ PCRCloning System. In addition, using the methods and assays described inthis Example, one of ordinary skill can, using only routineexperimentation, design and use analogous primer-adapters based on orcontaining other recombination sites or fragments thereof, such as attL,attR, attP, lox, FRT, etc.

Example 21 Mutational Analysis of the Bacteriophage Lambda attL and attRSites: Determinants of Att Site Specificity In Site-SpecificRecombination

To investigate the determinants of att site specificity, thebacteriophage lambda attL and attR sites were systematicallymutagenized. As noted herein, the determinants of specificity havepreviously been localized to the 7 bp overlap region (TTTATAC, which isdefined by the cut sites for the integrase protein and is the regionwhere strand exchange takes place) within the 15 bp core region(GCTTTTTTATACTAA) (SEQ ID NO:58) which is identical in all four lambdaatt sites, attB, attP, attL and attR. This core region, however, has notheretofore been systematically mutagenized and examined to defineprecisely which mutations produce unique changes in att sitespecificity.

Therefore, to examine the effect of att sequence on site specificity,mutant attL and attR sites were generated by PCR and tested in an invitro site-specific recombination assay. In this way all possible singlebase pair changes within the 7 bp overlap region of the core att sitewere generated as well as five additional changes outside the 7 bpoverlap but within the 15 bp core att site. Each attL PCR substrate wastested in the in vitro recombination assay with each of the attR PCRsubstrates.

Methods

To examine both the efficiency and specificity of recombination ofmutant attL and attR sites, a simple in vitro site-specificrecombination assay was developed. Since the core regions of attL andattR lie near the ends of these sites, it was possible to incorporatethe desired nucleotide base changes within PCR primers and generate aseries of PCR products containing mutant attL and attR sites. PCRproducts containing attL and attR sites were used as substrates in an invitro reaction with GATEWAY™ LR Clonase™ Enzyme Mix (Life Technologies,Inc.; Rockville, Md.). Recombination between a 1.5 kb attL PCR productand a 1.0 kb attR PCR product resulted in a 2.5 kb recombinant moleculethat was monitored using agarose gel electrophoresis and ethidiumbromide staining.

Plasmid templates pEZC1301 (FIG. 84) and pEZC1313 (FIG. 85), eachcontaining a single wild type attL or attR site, respectively, were usedfor the generation of recombination substrates. The following list showsprimers that were used in PCR reactions to generate the attL PCRproducts that were used as substrates in L×R Clonase reactions (capitalletters represent changes from the wild-type sequence, and the underlinerepresents the 7 bp overlap region within the 15 bp core att site; asimilar set of PCR primers was used to prepare the attR PCR productscontaining matching mutations):

GATEWAY™ sites (note: attL2 sequence in GATEWAY™ plasmids begins “accca”while the attL2 site in this example begins “agcct” to reflect wild-typeattL outside the core region.):

attL1:  (SEQ ID NO: 36) gggg agcct gcttttttGtacAaa gttggcatta taaaaa-agca ttgc  attL2:  (SEQ ID NO: 37)gggg agcct gctttCttGtacAaa gttggcatta taaaaa- agca ttgc  Wild-type:attL0:  (SEQ ID NO: 59) gggg agcct gcttttttatactaa gttggcatta taaaaa-agca ttgc  Single base changes from wild-type: attLT1A:  (SEQ ID NO: 60)gggg agcct gctttAttatactaa gttggcatta taaaaa- agca ttgc  attLT1C: (SEQ ID NO: 61) gggg agcct gctttCttatactaa gttggcatta taaaaa- agca ttgc attLT1G:  (SEQ ID NO: 62) gggg agcct gctttGttatactaa gttggcatta taaaaa-agca ttgc  attLT2A:  (SEQ ID NO: 63)gggg agcct gcttttAtatactaa gttggcatta taaaaa- agca ttgc  attLT2C: (SEQ ID NO: 64) gggg agcct gcttttCtatactaa gttggcatta taaaaa- agca ttgc attLT2G:  (SEQ ID NO: 65) gggg agcct gcttttGtatactaa gttggcatta taaaa-aagca ttgc  attLT3A:  (SEQ ID NO: 66)gggg agcct gctttttAatactaa gttggcatta taaaa- aagca ttgc  attLT3C: (SEQ ID NO: 67) gggg agcct gctttttCatactaa gttggcatta taaaa- aagca ttgc attLT3G:  (SEQ ID NO: 68) gggg agcct gctttttGatactaa gttggcatta taaaa-aagca ttgc  attLA4C:  (SEQ ID NO: 69)gggg agcct gcttttttCtactaa gttggcatta taaaa- aagca ttgc  attLA4G: (SEQ ID NO: 70) gggg agcct gcttttttGtactaa gttggcatta taaaa- aagca ttgc attLA4T:  (SEQ ID NO: 71) gggg agcct gcttttttTtactaa gttggcatta taaaa-aagca ttgc  attLT5A:  (SEQ ID NO: 72)gggg agcct gcttttttaAactaa gttggcatta taaaa- aagca ttgc  attLT5C: (SEQ ID NO: 73) gggg agcct gcttttttaCactaa gttggcatta taaaa- aagca ttgc attLT5G:  (SEQ ID NO: 74) gggg agcct gcttttttaGactaa gttggcatta taaaa-aagca ttgc  attLA6C:  (SEQ ID NO: 75)gggg agcct gcttttttatCctaa gttggcatta taaaa- aagca ttgc  attLA6G: (SEQ ID NO: 76) gggg agcct gcttttttatGctaa gttggcatta taaaa- aagca ttgc attLA6T:  (SEQ ID NO: 77) gggg agcct gcttttttatTctaa gttggcatta taaaa-aagca ttgc  attLC7A:  (SEQ ID NO: 78)gggg agcct gcttttttataAtaa gttggcatta taaaa- aagca ttgc  attLC7G: (SEQ ID NO: 79) gggg agcct gcttttttataGtaa gttggcatta taaaa- aagca ttgcattLC7T:  (SEQ ID NO: 80) gggg agcct gcttttttataTtaa gttggcatta taaaa-aagca ttgc  Single base changes outside of the 7 bp overlap: attL8:(SEQ ID NO: 81) gggg agcct Acttttttatactaa gttggcatta taaaa- aagca ttgc attL9:  (SEQ ID NO: 82) gggg agcct gcCtttttatactaa gttggcatta taaaaa-agca ttgc  attL10:  (SEQ ID NO: 83)gggg agcct gcttCtttatactaa gttggcatta taaaaa- agca ttgc  attL14: (SEQ ID NO: 84) gggg agcct gcttttttatacCaa gttggcatta taaaaa- agca ttgc attL15:  (SEQ ID NO: 85) gggg agcct gcttttttatactaG gttggcatta taaaaa-agca ttgc Note: additional vectors wherein the first nine bases are gggg agcca(i.e., substituting an adenine for the thymine in the positionimmediately preceding the 15-bp core region), which may or may notcontain the single base pair substitutions (or deletions) outlinedabove, can also be used in these experiments.

Recombination reactions of attL- and attR-containing PCR products wasperformed as follows:

8 μl of H₂0

2 μl of attL PCR product (100 ng)

2 μl of attR PCR product (100 ng)

4 μl of 5× buffer

4 μl of GATEWAY™ LR Clonase™ Enzyme Mix

20 μl total volume

Clonase reactions were incubated at 25° C. for 2 hours.2 μl of 10× Clonase stop solution (proteinase K, 2 mg/ml) were added tostop the reaction.10 μl were run on a 1% agarose gel.

Results

Each attL PCR substrate was tested in the in vitro recombination assaywith each of the attR PCR substrates. Changes within the first threepositions of the 7 bp overlap (TTTATAC) strongly altered the specificityof recombination. These mutant att sites each recombined as well as thewild-type, but only with their cognate partner mutant; they did notrecombine detectably with any other att site mutant. In contrast,changes in the last four positions (TTTATAC) only partially alteredspecificity; these mutants recombined with their cognate mutant as wellas wild-type att sites and recombined partially with all other mutantatt sites except for those having mutations in the first three positionsof the 7 bp overlap. Changes outside of the 7 bp overlap were found notto affect specificity of recombination, but some did influence theefficiency of recombination.

Based on these results, the following rules for att site specificitywere determined:

Only changes within the 7 bp overlap affect specificity.

Changes within the first 3 positions strongly affect specificity.

Changes within the last 4 positions weakly affect specificity.

Mutations that affected the overall efficiency of the recombinationreaction were also assessed by this method. In these experiments, aslightly increased (less than 2-fold) recombination efficiency withattLT1A and attLC7T substrates was observed when these substrates werereacted with their cognate attR partners. Also observed were mutationsthat decreased recombination efficiency (approximately 2-3 fold),including attLA6G, attL14 and attL15. These mutations presumably reflectchanges that affect Int protein binding at the core att site.

The results of these experiments demonstrate that changes within thefirst three positions of the 7 bp overlap (TTTATAC) strongly altered thespecificity of recombination (i.e., att sequences with one or moremutations in the first three thymidines would only recombine with theircognate partners and would not cross-react with any other att sitemutation). In contrast, mutations in the last four positions (TTTATAC)only partially altered specificity (i.e., att sequences with one or moremutations in the last four base positions would cross-react partiallywith the wild-type att site and all other mutant att sites, except forthose having mutations in one or more of the first three positions ofthe 7 bp overlap). Mutations outside of the 7 bp overlap were not foundto affect specificity of recombination, but some were found to influence(i.e., to cause a decrease in) the efficiency of recombination.

Example 22 Discovery of Att Site Mutations that Increase the CloningEfficiency of GATEWAY™ Cloning Reactions

In experiments designed to understand the determinants of att sitespecificity, point mutations in the core region of attL were made.Nucleic acid molecules containing these mutated attL sequences were thenreacted in an LR reaction with nucleic acid molecules containing thecognate attR site (i.e., an attR site containing a mutationcorresponding to that in the attL site), and recombinational efficiencywas determined as described above. Several mutations located in the coreregion of the att site were noted that either slightly increased (lessthan 2-fold) or decreased (between 2-4-fold) the efficiency of therecombination reaction (Table 3).

TABLE 3  Effects of attL mutations on Recombination Reactions. SEQEffect on Site ID NO: Sequence Recombination attL0 86 agcctgcttttttatactaagttggcatta attL5 87 agcctgctttAttata slightly ctaagttggcattaincreased attL6 88 agcctgcttttttata slightly Ttaagttggcatta increasedattL13 89 agcctgcttttttatG decreased ctaagttggcatta attL14 90agcctgcttttttata decreased cCaagttggcatta attL15 91 agcctgcttttttatadecreased ctaGgttggcatta con- 92 CAACTTnnTnnnAnnA sensus AGTTG

It was also noted that these mutations presumably reflected changes thateither increased or decreased, respectively, the relative affinity ofthe integrase protein for binding the core att site. A consensussequence for an integrase core-binding site (CAACTTNNT) has beeninferred in the literature but not directly tested (see, e.g., Ross andLandy, Cell 33:261-272 (1983)). This consensus core integrase-bindingsequence was established by comparing the sequences of each of the fourcore att sites found in attP and attB as well as the sequences of fivenon-att sites that resemble the core sequence and to which integrase hasbeen shown to bind in vitro. These experiments suggest that many moreatt site mutations might be identified which increase the binding ofintegrase to the core att site and thus increase the efficiency ofGATEWAY™ cloning reactions.

Example 23 Effects of Core Region Mutations on Recombination Efficiency

To directly compare the cloning efficiency of mutations in the att sitecore region, single base changes were made in the attB2 site of anattB1-TET-attB2 PCR product. Nucleic acid molecules containing thesemutated attB2 sequences were then reacted in a BP reaction with nucleicacid molecules containing non-cognate attP sites (i.e., wildtype attP2),and recombinational efficiency was determined as described above Thecloning efficiency of these mutant attB2 containing PCR productscompared to standard attB1-TET-attB2 PCR product are shown in Table 4.

TABLE 4  Efficiency of Recombination With Mutated attB2 Sites. SEQCloning Site ID NO: Sequence Mutation Efficiency attB0 93tcaagttagtataaaaaagcaggct attB1 94 ggggacaagttt gtacaaaaaagcaggct attB295 ggggaccacttt gtacaag aaagctgggt 100% attB2.1 96 ggggaAcacttt gtacaagaaagctgggt C→A  40% attB2.2 97 ggggacAacttt gtacaag aaagctgggt C→A 131%attB2.3 98 ggggaccCcttt gtacaag aaagctgggt A→C   4% attB2.4 99ggggaccaAttt gtacaag aaagctgggt C→A  11% attB2.5 100 ggggaccacGttgtacaag aaagctgggt T→G   4% attB2.6 101 ggggaccactGtgtacaag aaagctgggtT→G   6% attB2.7 102 ggggaccacttG gtacaag aaagctgggt T→G   1% attB2.8103 ggggaccacttt Ttacaag aaagctgggt G→T 0.5%

As noted above, a single base change in the attB2.2 site increased thecloning efficiency of the attB1-TET-attB2.2 PCR product to 131% comparedto the attB1-TET-attB2 PCR product. Interestingly, this mutation changesthe integrase core binding site of attB2 to a sequence that matches moreclosely the proposed consensus sequence.

Additional experiments were performed to directly compare the cloningefficiency of an attB1-TET-attB2 PCR product with a PCR product thatcontained attB sites containing the proposed consensus sequence (seeExample 22) of an integrase core binding site. The following attB siteswere used to amplify attB-TET PCR products:

attB1 (SEQ ID NO: 104) ggggacaagtttgtacaaaaaagcaggct attB1.6SEQ ID NO: 105) ggggacaaCtttgtacaaaaaagTTggct attB2 (SEQ ID NO: 106)ggggaccactttgtacaagaaagctgggt attB2.10 (SEQ ID NO: 107)ggggacAactttgtacaagaaagTtgggt

BP reactions were carried out between 300 ng (100 fmoles) of pDONR201(FIG. 49A) with 80 ng (80 fmoles) of attB-TET PCR product in a 20 μlvolume with incubation for 1.5 hrs at 25° C., creating pENTR201-TETEntry clones. A comparison of the cloning efficiencies of theabove-noted attB sites in BP reactions is shown in Table 5.

TABLE 5 Cloning efficiency of BP Reactions. Fold PCR product CFU/mlIncrease B1-tet-B2 7,500 B1.6-tet-B2 12,000 1.6 x B1-tet-B2.10 20,9002.8 x B1.6-tet-B2.10 30,100 4.0 x

These results demonstrate that attB PCR products containing sequencesthat perfectly match the proposed consensus sequence for integrase corebinding sites can produce Entry clones with four-fold higher efficiencythan standard Gateway attB1 and attB2 PCR products.

The entry clones produced above were then transferred to pDEST20 (FIG.40A) via LR reactions (300 ng (64 fmoles) pDEST20 mixed with 50 ng (77fmoles) of the respective pENTR201-TET Entry clone in 20 μl volume;incubated for 1 hr incubation at 25° C.). The efficiencies of cloningfor these reactions are compared in Table 6.

TABLE 6 Cloning Efficiency of LR Reactions. Fold pENTR201-TET x pDRST20CFU/ml Increase L1-tet-L2 5,800 L1.6-tet-L2 8,000 1.4 L1-tet-L2.1010,000 1.7 L1.6-tet-L2.10 9,300 1.6

These results demonstrate that the mutations introduced into attB1.6 andattB2.10 that transfer with the gene into entry clones slightly increasethe efficiency of LR reactions. Thus, the present invention encompassesnot only mutations in attB sites that increase recombination efficiency,but also to the corresponding mutations that result in the attL sitescreated by the BP reaction.

To examine the increased cloning efficiency of the attB1.6-TET-attB2.10PCR product over a range of PCR product amounts, experiments analogousto those described above were performed in which the amount of attB PCRproduct was titrated into the reaction mixture. The results are shown inTable 7.

TABLE 7 Titration of attB PCR products. Amount of attB PCR Fold product(ng) PCR product CFU/ml Increase 20 attB1-TET-attB2 3,500 6.1attB1.6-TET-attB2.10 21,500 50 attB1-TET-attB2 9,800 5.0attB1.6-TET-attB2.10 49,000 100 attB1-TET-attB2 18,800 2.8attB1.6-TET-attB2.10 53,000 200 attB1-TET-attB2 19,000 2.5attB1.6-TET-attB2.10 48,000

These results demonstrate that as much as a six-fold increase in cloningefficiency is achieved with the attB1.6-TET-attB2.10 PCR product ascompared to the standard attB1-TET-attB2 PCR product at the 20 ngamount.

Example 24 Determination of attB Sequence Requirements for OptimumRecombination Efficiency

To examine the sequence requirements for attB and to determine whichattB sites would clone with the highest efficiency from populations ofdegenerate attB sites, a series of experiments was performed. DegeneratePCR primers were designed which contained five bases of degeneracy inthe B-arm of the attB site. These degenerate sequences would thustransfer with the gene into Entry clone in BP reactions and subsequentlybe transferred with the gene into expression clones in LR reactions. Thepopulations of degenerate attB and attL sites could thus be cycled fromattB to attL back and forth for any number of cycles. By altering thereaction conditions at each transfer step (for example by decreasing thereaction time and/or decreasing the concentration of DNA) the reactioncan be made increasingly more stringent at each cycle and thus enrichfor populations of attB and attL sites that react more efficiently.

The following degenerate PCR primers were used to amplify a 500 bpfragment from pUC18 which contained the lacZ alpha fragment (only theattB portion of each primer is shown):

attB1 (SEQ ID NO: 108) GGGG ACAAGTTTGTACAAA AAAGC AGGCT attB1n16-20(SEQ ID NO: 109) GGGG ACAAGTTTGTACAAA nnnnn-AGGCT attB1n21-25(SEQ ID NO: 110) GGGG ACAAGTTTGTACAAA AAAGC-nnnnn attB 2(SEQ ID NO: 111) GGGG ACCACTTTGTACAAG AAAGC TGGGT attB2n16-20(SEQ ID NO: 112) GGGG ACCACTTTGTACAAG nnnnn-TGGGT attB2n21-25(SEQ ID NO: 113) GGGG ACCACTTTGTACAAG AAAGC-nnnnnThe starting population size of degenerate att sites is 4⁵ or 1024molecules. Four different populations were transferred through two BPreactions and two LR reactions. Following transformation of eachreaction, the population of transformants was amplified by growth inliquid media containing the appropriate selection antibiotic. DNA wasprepared from the population of clones by alkaline lysis miniprep andused in the next reaction. The results of the BP and LR cloningreactions are shown below.

BP-1, overnight reactions cfu/ml percent of control attB1-LacZa-attB278,500 100%  attB1n16-20-LacZa-attB2 1,140 1.5% attB1n21-25-LacZa-attB211,100  14% attB1-LacZa-attB2n16-20 710 0.9% attB1-LacZa-attB2n21-2516,600  21%

LR-1, pENTR201-LacZa x pDEST20/EcoRI, 1 hr reactions cfu/ml percent ofcontrol attL1-LacZa-attL2 20,000 100%  attL1n16-20-LacZa-attL2 2,125 11%attL1n21-25-LacZa-attL2 2,920 15% attL1-LacZa-attL2n16-20 3,190 16%attL1-LacZa-attL2n21-25 1,405  7%

BP-2, pEXP20-LacZa/ScaI x pDONR 201, 1 hr reactions cfu/ml percent ofcontrol attB1-LacZa-attB2 48,600 100%  attB1n16-20-LacZa-attB2 22,80047% attB1n21-25-LacZa-attB2 31,500 65% attB1-LacZa-attB2n16-20 42,40087% attB1-LacZa-attB2n21-25 34,500 71%

LR-2, pENTR201-LacZa x pDEST6/NcoI, 1 hr reactions cfu/ml percent ofcontrol attL1-LacZa-attL2 23,000 100% attL1n16-20-LacZa-attL2 49,000213% attL1n21-25-LacZa-attL2 18,000  80% attL1-LacZa-attL2n16-20 37,000160% attL1-LacZa-attL2n21-25 57,000 250%

These results demonstrate that at each successive transfer, the cloningefficiency of the entire population of att sites increases, and thatthere is a great deal of flexibility in the definition of an attB site.Specific clones may be isolated from the above reactions, testedindividually for recombination efficiency, and sequenced. Such newspecificities may then be compared to known examples to guide the designof new sequences with new recombination specificities. In addition,based on the enrichment and screening protocols described herein, one ofordinary skill can easily identify and use sequences in otherrecombination sites, e.g., other att sites, lox, FRT, etc., that resultin increased specificity in the recombination reactions using nucleicacid molecules containing such sequences.

Example 25 Design of att Site PCR Adapter-Primers

Additional studies were performed to design gene-specific primers with12 bp of attB1 and attB2 at their 5′-ends. The optimal primer design foratt-containing primers is the same as for any PCR primers: thegene-specific portion of the primers should ideally have a Tm of >50° C.at 50 mM salt (calculation of Tm is based on the formula 59.9+41(%GC)−675/n).

Primers: 12 bp attB1 (SEQ ID NO: 114): AA AAA GCA GGC TNN-forward gene-specific primer12 bp attB2 (SEQ ID NO: 115): A GAA AGC TGG GTN-reverse gene-specific primerattB1 adapter primer (SEQ ID NO: 116):  GGGGACAAGTTTGTACAAAAAA-GCAGGCTattB2 adapter primer (SEQ ID NO: 117):  GGGGACCACTTTGTACAAGAAA-GCTGGGT

Protocol:

(1) Mix 200 ng of cDNA library or 1 ng of plasmid clone DNA(alternatively, genomic DNA or RNA could be used) with 10 pmoles of genespecific primers in a 50 μl PCR reaction, using one or more polypeptideshaving DNA polymerase activity such as those described herein. (Theaddition of greater than 10 pmoles of gene-specific primers can decreasethe yield of attB PCR product. In addition, if RNA is used, a standardreverse transcriptase-PCR (RT-PCR) protocol should be followed; see,e.g., Gerard, G. F., et al., FOCUS 11:60 (1989); Myers, T. W., andGelfand, D. H., Biochem. 30:7661 (1991); Freeman, W. N., et al.,BioTechniques 20:782 (1996); and U.S. application Ser. No. 09/064,057,filed Apr. 22, 1998, the disclosures of all of which are incorporatedherein by reference.)

1^(st) PCR Profile:

(a) 95° C. for 3 minutes(b) 10 cycles of:

(i) 94° C. for 15 seconds

(ii) 50° C.* for 30 seconds *The optimal annealing temperature isdetermined by the calculated Tm of the gene-specific part of the primer.

(iii) 68° C. for 1 minute/kb of target amplicon

(c) 68° C. for 5 minutes(d) 10° C. hold

(2) Transfer 10 μl to a 40 μl PCR reaction mix containing 35 pmoles eachof the attB1 and attB2 adapter primers.

PCR Profile:

(a) 95° C. for 1 minute(b) 5 cycles of:

(i) 94° C. for 15 seconds

(ii) 45° C.* for 30 seconds *The optimal annealing temperature isdetermined by the calculated Tm of the gene-specific part of the primer.

(iii) 68° C. for 1 minute/kb of target amplicon

(c) 15-20 cycles** of: ** 15 cycles is sufficient for low complexitytargets.

(i) 94° C. for 15 seconds

(ii) 55° C.* for 30 seconds *The optimal annealing temperature isdetermined by the calculated Tm of the gene-specific part of the primer.

(iii) 68° C. for 1 minute/kb of target amplicon

(d) 68° C. for 5 minutes(e) 10° C. hold

Notes:

-   1. It is useful to perform a no-adapter primer control to assess the    yield of attB PCR product produced.-   2. Linearized template usually results in slightly greater yield of    PCR product.

Example 26 One-Tube Recombinational Cloning Using the GATEWAY™ CloningSystem

To provide for easier and more rapid cloning using the GATEWAY™ cloningsystem, we have designed a protocol whereby the BP and LR reactions maybe performed in a single tube (a “one-tube” protocol). The following isan example of such a one-tube protocol; in this example, an aliquot ofthe BP reaction is taken before adding the LR components, but the BP andLR reactions may be performed in a one-tube protocol without firsttaking the BP aliquot:

Reaction Component Volume attB DNA (100-200 ng/25 μl reaction) 1-12.5μl    attP DNA (pDONR201) 150 ng/μl 2.5 μl 5X BP Reaction Buffer 5.0 μlTris-EDTA (to 20 μl)  BP Clonase 5.0 μl Total vol.  25 μl

After the above components were mixed in a single tube, the reactionmixtures were incubated for 4 hours at 25° C. A 5 μl aliquot of reactionmixture was removed, and 0.5 μl of 10× stop solution was added to thisreaction mixture and incubated for 10 minutes at 37° C. Competent cellswere then transformed with 1-2 μl of the BP reaction per 100 μl ofcells; this transformation yielded colonies of Entry Clones forisolation of individual Entry Clones and for quantitation of the BPReaction efficiency.

To the remaining 20 μl of BP reaction mixture, the following componentsof the LR reaction were added:

Reaction Component Final Concentration Volume Added NaCl 0.75M 1 μlDestination Vector 150 ng/ul 3 μl LR Clonase 6 μl Total vol. 30 μl 

After the above components were mixed in a single tube, the reactionmixtures were incubated for 2 hours at 25° C. 3 μl of 10× stop solutionwas added, and the mixture was incubated for 10 minutes at 37° C.Competent cells were then transformed with 1-2 μl of the reactionmixture per 100 μl of cells

Notes:

-   1. If desired, the Destination Vector can be added to the initial BP    reaction.-   2. The reactions can be scaled down by 2×, if desired.-   3. Shorter incubation times for the BP and/or LR reactions can be    used (scaled to the desired cloning efficiencies of the reaction),    but a lower number of colonies will typically result.-   4. To increase the number of colonies obtained by several fold,    incubate the BP reaction for 6-20 hours and increase the LR reaction    to 3 hours. Electroporation also works well with 1-2 ul of the    PK-treated reaction mixture.-   5. PCR products greater than about 5 kb may show significantly lower    cloning efficiency in the BP reaction. In this case, we recommend    using a one-tube reaction with longer incubation times (e.g., 6-18    hours) for both the BP and LR steps.

Example 27 Relaxation of Destination Vectors During the LR Reaction

To further optimize the LR Reaction, the composition of the LR Reactionbuffer was modified from that described above and this modified bufferwas used in a protocol to examine the impact of enzymatic relaxation ofDestination Vectors during the LR Reaction.

LR Reactions were set up as usual (see, e.g., Example 6), except that5×BP Reaction Buffer (see Example 5) was used for the LR Reaction. Toaccomplish Destination Vector relaxation during the LR Reaction,Topoisomerase I (Life Technologies, Inc., Rockville, Md.; Catalogue No.38042-016) was added to the reaction mixture at a final concentration of˜15U per μg of total DNA in the reaction (for example, for reactionmixtures with a total of 400 ng DNA in the 20 μl LR Reaction, ˜6 unitsof Topoisomerase I was added). Reaction mixtures were set up as follows:

Reaction Component Volume ddH₂O 6.5 μl  4X BP Reaction Buffer 5 μl 100ng single chain/linear pENTR CAT, 50 ng/μl 2 μl 300 ng singlechain/linear pDEST6, 150 ng/μl 2 μl Topoisomerase I, 15 U/ml 0.5 μl  LRClonase 4 μl

Reaction mixtures were incubated at 25° C. for 1 hour, and 2 μl of 2μg/μl Proteinase K was then added and mixtures incubated for 10 minutesat 37° C. to stop the LR Reaction. Competent cells were then transformedas described in the preceding examples. The results of these studiesdemonstrated that relaxation of substrates in the LR reaction usingTopoisomerase I resulted in a 2- to 10-fold increase in colony outputcompared to those LR reactions performed without including TopoisomeraseI.

Having now fully described the present invention in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious to one of ordinary skill in the art that the same can beperformed by modifying or changing the invention within a wide andequivalent range of conditions, formulations and other parameterswithout affecting the scope of the invention or any specific embodimentthereof, and that such modifications or changes are intended to beencompassed within the scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

1. An isolated nucleic acid molecule comprising a nucleotide sequenceselected from the group of nucleotide sequences consisting of an attB1nucleotide sequence as set forth in FIG. 9, an attB2 nucleotide sequenceas set forth in FIG. 9, an attP1 nucleotide sequence as set forth inFIG. 9, an attP2 nucleotide sequence as set forth in FIG. 9, an attL1nucleotide sequence as set forth in FIG. 9, an attL2 nucleotide sequenceas set forth in FIG. 9, an attR1 nucleotide sequence as set forth inFIG. 9, an attR2 nucleotide sequence as set forth in FIG. 9, apolynucleotide complementary thereto, and a mutant, fragment, orderivative thereof. 2-9. (canceled)
 10. The isolated nucleic acidmolecule of claim 1, further comprising one or more functional orstructural nucleotide sequences selected from the group consisting ofone or more multiple cloning sites, one or more localization signals,one or more transcription termination sites, one or more transcriptionalregulatory sequences, one or more translational signals, one or moreorigins of replication, one or more fusion partner peptide-encodingnucleic acid molecules, one or more protease cleavage sites, and one ormore 5′ polynucleotide extensions.
 11. The nucleic acid molecule ofclaim 10, wherein said transcriptional regulatory sequence is apromoter, an enhancer, or a repressor.
 12. The nucleic acid molecule ofclaim 10, wherein said fusion partner peptide-encoding nucleic acidmolecule encodes glutathione S-transferase (GST), hexahistidine (HiS₆)or thioredoxin (Trx).
 13. The nucleic acid molecule of claim 10, whereinsaid 5′ polynucleotide extension consists of from one to five nucleotidebases.
 14. The nucleic acid molecule of claim 13, wherein said 5′polynucleotide extension consists of four or five guanine nucleotidebases.
 15. A primer nucleic acid molecule suitable for amplifying atarget nucleotide sequence, comprising the isolated nucleic acidmolecule of claim for a portion thereof linked to a target-specificnucleotide sequence useful in amplifying said target nucleotidesequence.
 16. The primer nucleic acid molecule of claim 15, wherein saidprimer comprises an attB1 nucleotide sequence having the sequence shownin FIG. 9 or a portion thereof, or a polynucleotide complementary to thesequence shown in FIG. 9 or a portion thereof.
 17. The primer nucleicacid molecule of claim 15, wherein said primer comprises an attB2nucleotide sequence having the sequence shown in FIG. 9 or a portionthereof, or a polynucleotide complementary to the sequence shown in FIG.9 or a portion thereof.
 18. The primer nucleic acid molecule of claim15, further comprising a 5′ terminal extension of four or five guaninebases.
 19. A vector comprising the isolated nucleic acid molecule ofclaim
 1. 20. The vector of claim 19, wherein said vector is anExpression Vector.
 21. (canceled)
 22. A method of synthesizing oramplifying one or more nucleic acid molecules comprising: (a) mixing oneor more nucleic acid templates with at least one polypeptide havingpolymerase or reverse transcriptase activity and at least a first primercomprising a template-specific sequence that is complementary to orcapable of hybridizing to said templates and at least a second primercomprising all or a portion of a recombination site wherein said atleast a portion of said second primer is homologous to or complementaryto at least a portion of said first primer; and (b) incubating saidmixture under conditions sufficient to synthesize or amplify one or morenucleic acid molecules complementary to all or a portion of saidtemplates and comprising one or more recombination sites or portionsthereof at one or both termini of said molecules. 23-25. (canceled) 26.An isolated nucleic acid molecule comprising one or more attrecombination sites comprising at least one mutation in its core regionthat increases the specificity of interaction between said recombinationsite and a second att recombination site.
 27. The isolated nucleic acidmolecule of claim 26, wherein said mutation is at least one substitutionmutation of at least one nucleotide in the seven basepair overlap regionof said core region of said recombination site.
 28. The isolated nucleicacid molecule of claim 26, wherein said nucleic acid molecule comprisesthe sequence NNNATAC, wherein “N” refers to any nucleotide with theproviso that if one of the first three nucleotides in the consensussequence is a TIU, then at least one of the other two of the first threenucleotides is not a TIU.
 29. An isolated nucleic acid moleculecomprising one or more mutated att recombination sites comprising atleast one mutation in its core region that enhances the efficiency ofrecombination between a first nucleic acid molecule comprising saidmutated att recombination site and a second nucleic acid moleculecomprising a second recombination site that interacts with said mutatedatt recombination site.
 30. The isolated nucleic acid molecule of claim29, wherein said mutated att recombination site is a mutated attL sitecomprising a core region having the nucleotide sequencecaacttnntnnnannaagttg (SEQID NO:92), wherein “n” represents anynucleotide.
 31. The isolated nucleic acid molecule of claim 30, whereinsaid mutated attL recombination site comprises a core region having anucleotide sequence selected from agcctgctttattatactaagttggcatta (attL5;SEQ ID NO:87) and agcctgcttttttatattaagttggcatta (attL6; SEQ ID NO:88).32. The isolated nucleic acid molecule of claim 29, wherein said mutatedatt recombination site comprises a core region having a nucleotidesequence selected from the group consisting ofggggacaactttgtacaaaaaagttggct (attB1.6; SEQ ID NO:105),ggggacaactttgtacaagaaagctgggt (attB2.2; SEQ ID NO:97), andggggacaactttgtacaagaaagttgggt (attB2.10; SEQ ID NO: 107). 33-38.(canceled)